Sebastian Rudolph was born in Berlin in 1968 to actress Hildegard Schmahl and director Niels-Peter Rudolph. From 1989 to 1990 he trained to be an actor at the Schauspielstudio Frese in Hamburg. In the following years, he appeared on stage in various theaters, among them the Theater Basel (Switzerland), the Berliner Ensemble, the Schaubühne Berlin, the Deutsches Theater Berlin, the Volksbühne Berlin, the Schauspielhaus Hamburg and Vienna's Burgtheater. Productions he starred in range from "Leonce and Lena" to "Drei Schwestern" ("Three Sisters") and "Heinrich IV" ("Henry IV") to Karmakar's "Die Nacht singt ihre Lieder" ("Nightsongs"). Christoph Marthaler, Luc Bondy, Robert Wilson, Christoph Schlingensief and Schorsch Kamerun are just a few of the renowned directors, Rudolph has worked with.

In addition to his work in theater, Rudolph has also been starring in film and TV productions. His first role in a feature film was the lead role in Peter Timm's comedy "Manta – Der Film" (1991), in which he played a 17-year-old student driver. Following this, he starred in a major role in Joseph Vilsmaier's war drama "Stalingrad" (1992). Except for a minor appearance as an officer in "The English Patient" (US/GB 1996), in the following years Rudolph worked almost exclusively for TV.

He starred alongside Franka Potente in the gay-straight love comedy "Coming In" (1997) and alongside Jürgen Vogel in Bernd Schadewald's junkie drama "Der Pirat" (1998). In the comedy "Der Millionär und die Stripperin" (2001), he portrayed the son of a rich, enamored business man, while director Markus Imboden cast him as murder suspect for his crime film "Mörderische Erpressung" (2006). In 2008, Rudolph played the author Axel Eggebrecht in the biopic "Remarque - Sein Weg zum Ruhm". Often, Rudolph also guest starred in TV series such as "Tatort", "Wolffs Revier", "Alphateam - Die Lebensretter im OP" and "Doppelter Einsatz".

In the 2009/2010 season, Rudolph joined the Thalia Theater ensemble in Hamburg and was elected Actor of the Year in 2012 by the theater magazine 'Theater heute' for his much-acclaimed performance in "Faust I+II".

Despite his on-stage work, he also kept appearing in front of the camera, like in Markus Imboden's "Tod einer Brieftaube" (2012, TV), in which he played a kidnap victim. He starred in another important role in Roland Suso Richter's "Die Spiegel-Affäre", portraying the 'Spiegel' magazine editor Rudolf Augstein. Sherry Hormann cast him for her political conspiracy thriller "Tödliche Geheimnisse" ("Deadly Leaks", 2016) in a supporting role as an idealistic editorial journalist.

On the big screen, Rudolph starred in smaller roles in the adolescence drama "4 Könige" ("4 Kings", 2015) and in the relationship drama "Gleißendes Glück" ("Original Bliss", 2016). In the award-winning "Whatever Happens Next" (DE/PL 2018), he played the lead role of a father, who leaves his family to live as a friendly good-for-nothing and impostor.

He was then seen in a supporting role in Florian Henckel von Donnersmarck's artist biography "Werk ohne Autor" ("Never Look Away", 2918) and as a sensitive neurologist in Katrin Gebbe's award-winning mystery-horror "Pelikanblut" ("Pelican Blood", 2019). He played a leading role in Ulrich Köhler and Henner Winckler's "Das freiwillige Jahr" ("A Voluntary Year", 2019), about a high school graduate (Maj-Britt Klenke) in provincial Germany who wants to emancipate herself from her single father (Rudolph). He was nominated for the German Film Critics Award for this role. He also appeared as the father of the female lead in Maggie Peren's romantic comedy "Hello Again - Ein Tag für immer" ("Hello Again - A Wedding A Day", 2020).  

On television, Rudolph took on (supporting) roles in the Siegfried Lenz adaptation "Deutschstunde" ("The German Lesson", 2019), the family saga "Altes Land" (2020) and an episode of the series "Der Zürich-Krimi" (2021) with Christian Kohlund, among others. In the streaming series "Wir Kinder vom Bahnhof Zoo" ("We Children From Bahnhof Zoo", 2021), he played a masochistic client of a prostitute.  

Sebastian Rudolph had another starring role in the award-winning drama "Piaffe" (2022), as a botanist who begins a sadomasochistic affair with a soundmaker who grows a tail out of her coccyx while she imitates the sounds of a horse.