A fibrinolytic enzyme producing bacterium was isolated and identified as Bacillus subtilis A26 on the basis of the 16S rRNA gene sequence. The fibrin zymography analysis reveals the presence of at least three fibrinolytic enzymes. The crude enzyme exhibited maximal activity at 60 °C and pH 8.0. Medium composition and culture conditions for the enzyme production by B. subtilis A26 were optimized using two statistical methods. The Plackett–Burman statistical design was applied to find the key ingredients and conditions for the best yield of enzyme production. Five significant variables (hulled grain of wheat, casein peptone, NaCl, CaCl₂, and initial pH) were selected for the optimization studies. The response surface methodological approach was used to determine the optimal concentrations and conditions. The optimized medium contained 40.0 g·L^–1hulled grain of wheat, 3.53 g·L^–1casein peptone, 4.0 g·L^–1CaCl₂, 3.99 g·L^–1NaCl, 0.01 g·L^–1MgSO₄, and 0.01 g·L^–1KH₂PO₄, pH 7.78. The medium optimization resulted in a 4.2-fold increased level of fibrinolytic production (269.36 U·mL^–1) compared with that obtained with the initial medium (63.45 U·mL^–1). A successful and significant improvement in the production of protease by the A26 strain was accomplished using inexpensive carbon substrate (hulled grain of wheat), allowing a significant reduction in the cost of medium constituents.