D'Ambrosio began competitive karting in 1999, winning three consecutive Belgian kart championships in the Mini class (1999), Junior class (2000), and Formula A (2002). He also won the Junior Monaco Kart Cup in 2000 and claimed the World Cup Formula A championship in 2002 at age 16.
D'Ambrosio graduated to single-seaters in 2003 and immediately won the Belgian Formula Renault championship with five victories, driving for Thierry Boutsen Racing. That success earned him a place on the Renault F1 Driver Development Programme for 2004, when he competed in French Formula Renault 2.0 and finished fourth overall as the highest-placed rookie. He followed that with seasons in Italian Formula Renault before progressing to the Formula Renault 3.5 Series in 2006 with Tech 1 Racing, though he left that championship early to contest Euroseries 3000, finishing fifth.
In 2007, d'Ambrosio entered the inaugural International Formula Master championship with Cram Competition and dominated it, winning five races, scoring eleven podiums, and claiming seven fastest laps in sixteen races to become champion.
D'Ambrosio joined the GP2 Series in 2008 with DAMS, the primary feeder series to Formula One, and also raced in the GP2 Asia Series. He finished 11th in both championships in his debut year, collecting two podiums in each. In the 2008–09 Asia Series, he was runner-up behind Kamui Kobayashi. His 2010 GP2 campaign produced a breakthrough season: he won at Monaco for his first series victory, took a pole position at Spa-Francorchamps, and demonstrated consistent pace across the year.
On 21 December 2010, d'Ambrosio was confirmed as a race driver for Virgin Racing in the 2011 Formula One World Championship, partnering Timo Glock. He became the first Belgian driver to compete at the Belgian Grand Prix since Thierry Boutsen in 1993. Driving the largely uncompetitive Virgin MVR-02, he finished sixteen of nineteen races, with a best result of two 14th-place finishes in Australia and Canada, and ended the season 24th in the Drivers' Championship.
For 2012, d'Ambrosio was named reserve driver for Lotus F1, supporting Kimi Räikkönen and Romain Grosjean. He made his one race start for the team at the Italian Grand Prix, replacing Grosjean who had received a one-race ban for causing a multi-car collision at Spa. D'Ambrosio qualified 16th and finished 13th on race day, one lap down on winner Lewis Hamilton but a creditable performance in difficult circumstances. He continued as Lotus reserve driver for 2013.
D'Ambrosio joined Formula E from its inaugural season in 2014 with Dragon Racing, partnering Oriol Servià and Loïc Duval. He scored points on debut at the Beijing ePrix and won his first race at the 2015 Berlin ePrix, after initial winner Lucas di Grassi was disqualified for a technical violation. He followed that with back-to-back podiums at the London ePrix double-header and finished fourth in the championship. Dragon Racing finished second in the Teams' Championship that year.
He claimed his second win at the 2016 Mexico City ePrix — again after di Grassi was disqualified — and earned a first Formula E pole position at the 2015 Punta del Este ePrix. His time with Dragon Racing became increasingly difficult from the 2016–17 season, when the team built its own powertrains in partnership with Faraday Future before that relationship collapsed due to the startup's financial difficulties. He moved to Mahindra Racing for 2018–19 and won his third race at the 2019 Marrakesh ePrix, briefly leading the Drivers' Championship at the season midpoint. He retired from professional competition at the conclusion of the 2019–20 season, ending his Formula E career with 18th place overall in his final race in Berlin.
D'Ambrosio returned to Formula E immediately in a management role, joining ROKiT Venturi Racing as Deputy Team Principal for the 2020–21 season. He was promoted to Team Principal in November 2021, and under his leadership the team achieved its most successful campaign in the 2021–22 World Championship, winning five races, scoring ten podiums, and finishing second in the Teams' Championship with 295 points. He departed in September 2022 ahead of the team's transition to Maserati MSG Racing.
In 2023, d'Ambrosio joined Mercedes-AMG Petronas F1 Team in a formal capacity as Driver Development Director, overseeing Mercedes' junior driver programme. When team principal Toto Wolff underwent knee surgery, d'Ambrosio stood in as acting team representative at several Grand Prix weekends. In May 2024, Scuderia Ferrari announced d'Ambrosio would join as Deputy Team Principal and Head of the Ferrari Driver Academy, beginning 1 October 2024.
D'Ambrosio was born to Henri and Giselle d'Ambrosio. In 2013 he married American-Chilean model Natalie Sifferman; they divorced two years later. In 2020 he married Austrian jewellery designer Eleonore von Habsburg at the Civil Registry of Monaco. Their son Otto d'Ambrosio was born on 20 October 2021, named after Eleonore's grandfather Otto von Habsburg. A daughter, Zita d'Ambrosio, was born in August 2024, named after Zita von Habsburg.
D'Ambrosio also made a television appearance in the first episode of the Amazon motoring show The Grand Tour, serving as a test driver to establish lap times for the LaFerrari, Porsche 918, and McLaren P1.
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