[Objective] To optimize the fermentation conditions of Streptomyces strain MY0504 from marine environment and improve the fibrinolytic activity in fermentation broth. [Methods] Based on the growth curve of MY0504 and single factor test, the main factors affecting enzyme activity were found through the Plackett-Burman design, and the fermentation conditions were further optimized using steepest ascent experiment and Box-Behnken central composite design. [Results] The optimal fermentation conditions were as follows: glucose 21.68 g/L, yeast powder 25.31 g/L, NaCl 5.0 g/L, K2HPO4·3H2O 3.0 g/L, MgSO4·7H2O 0.5 g/L, FeSO4·7H2O 0.02 g/L, medium volume 50 mL (250 mL flask), inoculum size 10% (V/V), initial pH 7.5, fermentation temperature 24 °C, shaking speed 200 r/min, fermentation time 4.5 d. The enzyme activity reached 2 190.6 U/mL in fermentation broth. [Conclusion] The optimal fermentation conditions of strain MY0504 producing fibrinolytic enzyme were obtained, which lay a foundation for further purification and characterization of the fibrinolytic enzyme.