D'Ambrosio began karting in 1999, winning Belgian national titles in the Mini, Junior, and Formula A classes and claiming the World Cup Formula A championship in 2002. He graduated to single-seaters in 2003, winning the Belgian Formula Renault championship with Thierry Boutsen Racing. After seasons in French, Italian, and European Formula Renault series under the Renault F1 Driver Development Programme, d'Ambrosio won the inaugural International Formula Master championship in 2007 with five victories in sixteen races.
D'Ambrosio spent three seasons in the GP2 Series with the DAMS team from 2008 to 2010. He finished runner-up to Kamui Kobayashi in the 2008–09 GP2 Asia Series and had a breakout 2010 GP2 season, taking his first victory at Monaco and his first pole position at Spa-Francorchamps.
D'Ambrosio was named reserve driver for the Renault F1 Team in January 2010 and made four practice appearances with Virgin Racing later that year. On 21 December 2010, he was confirmed as a race driver for Virgin for the 2011 Formula One World Championship, replacing Lucas di Grassi alongside Timo Glock. He finished sixteen of nineteen races, achieving two 14th-place finishes as his best results, and became the first Belgian driver to compete at the Belgian Grand Prix since Thierry Boutsen in 1993.
For 2012, d'Ambrosio served as official reserve driver for Lotus F1, supporting Kimi Räikkönen and Romain Grosjean. He replaced Grosjean at the Italian Grand Prix after the Frenchman received a one-race ban, qualifying 16th and finishing 13th. He continued as Lotus reserve driver through 2013.
D'Ambrosio joined Dragon Racing for the inaugural 2014–15 Formula E season, partnering Oriol Servià and Loïc Duval. He scored points on debut at the Beijing ePrix and claimed his first victory at the 2015 Berlin ePrix after initial winner Lucas di Grassi was disqualified for a technical infringement. He finished fourth in the championship that season, the only driver to complete every racing lap without missing the top ten more than twice.
He remained with Dragon Racing for three further seasons. His second victory came at the 2016 Mexico City ePrix, again after di Grassi's disqualification. During the 2017–18 season he returned to the podium at the Zürich ePrix.
In October 2018 d'Ambrosio joined Mahindra Racing. He secured his third career victory at the 2019 Marrakesh ePrix and led the drivers' championship at the halfway point of the season before a difficult second half left him eleventh overall. He retired from professional driving at the end of the 2019–20 season, finishing 16th in the championship.
D'Ambrosio joined ROKiT Venturi Racing as Deputy Team Principal in October 2020 and was promoted to Team Principal in November 2021. Under his leadership the team achieved its most successful Formula E season in 2021–22, winning five races, scoring ten podiums, and finishing second in the World Teams' Championship with 295 points. He departed ahead of the team's transition to Maserati MSG Racing.
In 2023 d'Ambrosio took on a formal role as Driver Development Director at the Mercedes-AMG Petronas F1 Team, briefly deputising for Team Principal Toto Wolff at grands prix in Japan and Qatar while Wolff recovered from knee surgery.
In May 2024 Scuderia Ferrari announced d'Ambrosio would join as Deputy Team Principal and Head of the Ferrari Driver Academy from 1 October 2024, overseeing the team's young driver development programme.
D'Ambrosio's career arc — from a Formula Renault champion to a three-time Formula E race winner to a senior executive at one of motorsport's most storied teams — illustrates the breadth of the modern motorsport career. His Formula E record of completing every racing lap in his debut season and winning three ePrix highlighted consistent racecraft, while his management success at Venturi demonstrated leadership ability that attracted the interest of the sport's biggest constructors.