Bacillus sp. can biosynthesize fibrinolytic enzymes. Culture conditions and nutrient sources are important factors affecting microbial growth and production of the Fibrinolytic enzyme. Therefore, we investigated the culture conditions of the strain Bacillus sp. ES4, such as temperature, pH, C nutrient source (glucose, sucrose, glycerol, and maltose), N source (yeast extract, meat content, peptone, and tryptone), metal ions (Ca2+, Mg2+, Mn2+, Zn2+, K+, Fe2+, and Cu2+), and time to obtain enzyme product. The results show that for Bacillus sp. ES4, the best C source was glucose, peptone and yeast extract. Ca2+ and Mg2+ are nutritional factors that greatly influence the biosynthesis of the fibrinolytic enzyme of strain ES4. At the culture conditions of 370C and pH = 6.5, the strains for the best enzyme activity at 24 hours and the enzyme activity of the strain when cultured in a 250 ml flask containing 50 ml of medium for the highest enzyme activity at 450±25. FU/ml.