Abstract: In the current study, liquid fermentation medium and culture conditions were studied for fibrinolytic enzyme production from middle acidophilic Actinomycete sp.YY21 by single factor experiment.The results showed that the fermentation medium consisted of 1.7% of millet, 2% of glucose, 0.2% of calcium carbonate, 0.5% of sodium chloride and 0.6% of peptone.The optimized fermentation conditions were as follows: 50 m L liquid medium in 250 m L flasks, inoculation volume 5%, rotate speed 160 r/min, culture time 4 d and culture temperature 22 ℃. Under the optimized conditions, the degradation zone area of Actinomycete sp.YY21 product by liquid fermentation reached 164.38 mm² in the fibrin plate, increasing by 88% than that of original conditions ( 87.58 mm²) .Meanwhile, the fermentation time was decreased by 3 d.The production of fibrinolytic enzyme from middle acidophilic Actinomycete sp. YY21 was enhanced effectively by study of liquid fermentation medium and culture conditions.