Albert married first, to Princess Dorothea (1 August 1504 – 11 April 1547), daughter of King Frederick I of Denmark, in 1526. They had six children:

Turning to a more active life, Albert accompanied Emperor Maximilian I to Italy in 1508 and after his return spent some time in the Kingdom of Hungary.

Duke Frederick of Saxony, Grand Master of the Teutonic Order, died in December 1510. Albert was chosen as his successor early in 1511 in the hope that his relationship to his maternal uncle, Sigismund I the Old, Grand Duke of Lithuania and King of Poland, would facilitate a settlement of the disputes over eastern Prussia, which had been held by the order under Polish suzerainty since the Second Peace of Thorn (1466).

The new Grand Master, aware of his duties to the empire and to the papacy, refused to submit to the crown of Poland. As war over the order's existence appeared inevitable, Albert made strenuous efforts to secure allies and carried on protracted negotiations with Emperor Maximilian I. The ill-feeling, influenced by the ravages of members of the Order in Poland, culminated in a war which began in December 1519 and devastated Prussia. Albert was granted a four-year truce early in 1521.

The dispute was referred to Emperor Charles V and other princes, but as no settlement was reached Albert continued his efforts to obtain help in view of a renewal of the war. For this purpose he visited the Diet of Nuremberg in 1522, where he made the acquaintance of the Reformer Andreas Osiander, by whose influence Albert was won over to Protestantism.

After some delay Sigismund assented to the offer, with the provision that Prussia should be treated as a Polish fiefdom; and after this arrangement had been confirmed by a treaty concluded at Kraków, Albert pledged a personal oath to Sigismund I and was invested with the duchy for himself and his heirs on 10 February 1525.

In imperial politics Albert was fairly active. Joining the League of Torgau in 1526, he acted in unison with the Protestants, and was among the princes who banded and plotted together to overthrow Charles V after the issue of the Augsburg Interim in May 1548. For various reasons, however, poverty and personal inclination among others, he did not take a prominent part in the military operations of this period.

Albert was the first German noble to support Luther's ideas and in 1544 founded the University of Königsberg, the Albertina, as a rival to the Roman Catholic Cracow Academy. It was the second Lutheran university in the German states, after the University of Marburg.

A relief of Albert over the Renaissance-era portal of Königsberg Castle's southern wing was created by Andreas Hess in 1551 according to plans by Christoph Römer. Another relief by an unknown Artist was included in the wall of the Albertina's original campus. This depiction, which showed the duke with his sword over his shoulder, was the popular "Albertus", the symbol of the university. The original was moved to Königsberg Public Library to protect it from the elements, while the Sculptor Paul Kimritz created a duplicate for the wall. Another version of the "Albertus" by Lothar Sauer was included at the entrance of the Königsberg State and Royal Library.

After Osiander's death in 1552, Albert favoured a preacher named Johann Funck, who, with an adventurer named Paul Skalić, exercised great influence over him and obtained considerable wealth at public expense. The state of turmoil caused by these religious and political disputes was increased by the possibility of Albert's early death and the need, should that happen, to appoint a regent, as his only son, Albert Frederick was still a mere youth. The duke was forced to consent to a condemnation of the teaching of Osiander, and the climax came in 1566 when the Estates appealed to King Sigismund II Augustus of Poland, Albert's cousin, who sent a commission to Königsberg. Skalić saved his life by FLIGHT, but Funck was executed. The question of the regency was settled, and a form of Lutheranism was adopted and declared binding on all teachers and Preachers.

He married secondly to Anna Maria (1532–20 March 1568), daughter of Eric I, Duke of Brunswick-Lüneburg, in 1550. The couple had two children:

In 1880 Friedrich Reusch created a sandstone bust of Albert at the Regierungsgebäude, the administrative building for Regierungsbezirk Königsberg. On 19 May 1891 Reusch premiered a famous statue of Albert at Königsberg Castle with the inscription: "Albert of Brandenburg, Last Grand Master, First Duke in Prussia". Albert Wolff also designed an Equestrian statue of Albert located at the new campus of the Albertina. King's Gate contains a statue of Albert.

Albert was oft-honored in the quarter Maraunenhof in northern Königsberg. Its main street was named Herzog-Albrecht-Allee in 1906. Its town square, König-Ottokar-Platz, was renamed Herzog-Albrecht-Platz in 1934 to match its church, the Herzog-Albrecht-Gedächtniskirche.