Ružička was born in Vukovar (until 1920 in the Kingdom of Hungary, Austro-Hungarian Empire, today in Croatia). His family of craftsmen and farmers was mostly of Croat origin, with a Czech great-grandparent, Ružička, and a great-grandmother and a great-grandfather from Austria. He lost his father, Stjepan, at the age of four, and his mother, Amalija Sever, took him and his younger brother Stjepan, to live in Osijek.
Ružička attended the classics program secondary school in Osijek. He changed his original idea of becoming a priest and switched to studying technical disciplines. Chemistry was his choice, probably because he hoped to get a position at the newly opened sugar refinery built in Osijek.
Owing to the excessive hardship of everyday and political life, he left and chose the High Technical School in Karlsruhe in Germany. He was a good student in areas he liked and that he thought would be necessary and beneficial in the future, which was organic chemistry. That is why his physical chemistry professor, Fritz Haber (Nobel laureate in 1918), opposed his summa cum laude degree. However, in the course of his studies, Ružička set up excellent cooperation with Hermann Staudinger (a Nobel laureate in 1953). Studying within Staudingers department, he obtained his doctoral degree in 1910, then moving to Zürich as Staudingers assistant.