Helminthosporium gramineum Rabenh. f. sp. echinochloae (HGE), evaluated as a potential pathogenic fungus strain, could be applied to control barnyard grass in paddy field. For improving the weed control efficiency of HGE in paddy field, the barnyard grass was used as target weed and the adjuvants screened previously was designated as experimental factors. The uniform-precision rotatable central composite design was adopted to establish the regression equation of the lethal rate of barnyard grass in different formulations. The optimal formulation of HGE spore preparation containing 1.0 g spore powder, 0.25 mL soybean oil, 0.49 g carboxy methyl cellulose sodium (CMC), 0.10 mL SP-20 and synergists which dosage was 6.56 mg 10% Pyrazosulfuron-ethyl WP and 26 μL 2.5% Penoxsulam OD was achieved through the analysis of variance on the test results and the optimization of the mathematical model. These results provided useful formulation for next commercialized application of HGE.