Rudolf was the eldest son of the Saxon duke Albert II (c. 1250 – 1298), who initially ruled jointly with his brother John I but gradually concentrated on the Ascanian Saxe-Wittenberg territory.

Upon the death of Margrave Henry III of Meissen in 1288, Duke Albert II applied at his father-in-law King Rudolf for the enfeoffment of his son and heir with the Saxon County palatine on the Unstrut river, which ensued a long lasting dispute with the eager clan of the Wettin dynasty.

After Rudolf of Habsburg had died, Duke Albert II on 27 April 1292 wielded the Saxon electoral vote, electing Adolf of Nassau.

Still a minor when his father died on 25 August 1298, Rudolf I succeeded as Duke of Saxe-Wittenberg under the tutelage of his mother Agnes of Habsburg acting as guardian and regent.

When in February 1300, King Albert I tried to grant his eldest son Rudolf sole possession of Austria, the ecclesiastical prince-electors refused and a military conflict erupted.

Duke Rudolf I voted for Henry and also assisted him by providing money and troops, earning him the goodwill of the newly elected emperor.

For the first time, two candidates in the election claimed to have won it, the Wittelsbach duke Louis the Bavarian and Rudolf's Habsburg cousin Frederick the Fair.

After these and other sanctions by the Wittelsbach emperor, Duke Rudolf I decided to subordinate himself and his brother Wenceslas to Louis for tactical reasons and attempted to prove himself as a true support.

Thereafter, Louis finally changed his opinion of Rudolf and leased parts of the March of Lusatia, including the cities of Brietz, Fürstenwalde and Beeskow to him for a 12-year period.

His close ties to Charles IV were rewarded when he received the Altmark territory in 1347, whereby the Elbe river became the boundary between Saxony and Brandenburg.

Rudolf's greatest success came on 4 October 1355 when the emperor drafted the Golden Bull, the bulla aurea Saxonica, defining the future law of the Empire.

The Saxe-Wittenberg branch of the Ascanian dynasty became extinct with the death of Rudolf's grandson Duke Albert III in 1412, whereafter the Wittenberg estates and the Saxon electoral dignity passed to the Wettin margraves of Meissen.