He was the sixth (but fourth surviving) son of John VI, Prince of Anhalt-Zerbst, by his wife Sophie Auguste, daughter of Frederick III, Duke of Schleswig-Holstein-Gottorp.
Because he was still a minor, his mother, the Dowager Princess Sophie Auguste, acted as regent on behalf of him and his brothers.
In 1672, the sixteen-year-old John Louis began his Grand Tour with his older brother Karl William in Regensburg.
There they reunited with their middle brothers Anthony Günther and John Adolph, who had just returned from a journey to Italy.
During his stay at the Imperial court, John Louis and his brothers took the opportunity to make contacts and extend their knowledge.
He was appointed to go to Italy with the Hereditary Prince Augustus Frederick, eldest son and heir of Anthony Ulrich, Duke of Brunswick-Wolfenbüttel; although all necessary preparations were ready, the trip had to be cancelled because John Louis broke his leg.
During his stay in Vienna John Louis also made trips to Hungary, which belonged to the Habsburg Empire.
In the latter city he met Queen Marie-Thérèse of France, wife of King Louis XIV, who was extremely polite and accommodating to him.
He served in a regiment under the command of General von Scharfenberg and supervised an unsuccessful siege of a Hungarian fortress.
In Halle on 23 July 1687, John Louis married Christine Eleonore of Zeutsch (b. Hedersleben near Eisleben, 5 June 1666 – d. Dornburg, 17 May 1699), of an old Thuringian noble family.