[Objective] Response surface analysis was applied to optimize the fermentation conditions for producing fibrinolytic enzyme strain CNY16, and preliminarily studied its enzymology characteristics. [Methods] Through the Plackett-Burman design, the most significant effect on fibrinolytic enzyme production was obtained which include yeast extract, sodium chloride and revolving speed. The highest fibrinolytic activity area was investigated by the steepest climbing experiments, then, the optimal fermentation conditions of producing fibrinolytic enzyme strain CNY16 was analysised by Box-Behnken central composite design experiments. [Results] The optimal fermentation conditions of strain CNY16 was determined for 3.28% yeast extract, 1.14% sodium chloride, rotate speed at 166 r/min, the enzyme activity was 875.932 U/mL, the optimized enzyme activity was 46% higher than before. The optimum temperature for the fibrinolytic enzyme was 30 °C and the optimum pH was 6.5. [Conclusion] This experiment determined the most optimal fermentation conditions for producing fibrinolytic enzyme strain CNY16 and researched part of enzymatic properties, laied a foundation for further research and experiment of the the pilot study.