The two-time Olympic champion, set the world record for women's javelin with 72.28 metres in Stuttgart in 2008.
She recently participated at a Diamond League event in Zurich, where she came fifth.
"My body was clearly telling me that it was time to quit," Spotakova said.
Spotakova won her first gold at the Beijing Games in 2008 and defended her title four years later in London. She is one of two women to win more than one Olympic gold in javelin. In the 2016 Rio Games, she took bronze.
She also has won three World Championships and recently claimed bronze in the 2022 European Championships in August.
"Every fairy tale comes to an end, and mine had a wonderful happy ending in the form of a bronze medal at the European Championships in Munich, symbolically closing the circle," Spotakova said.