Abstract: The experiments with one factor and orthogonal design were conducted to optimize medium of antagonistic bacteria A3 strain against TMV (Pseudomonas putida) with mass growth (OD₆₀₀ value) as the index. A systematic study of carbon sources, nitrogen sources and inorganic chemicals was carried out. Carbon source “wheat bran”, nitrogen source “yeast extract powder” and inorganic chemical “CaCl₂, CoCl₂, MgSO₄, FeSO₄, KH₂PO₄”, were beneficial to the growth of A3. The results of L₁₈(3⁷) orthogonal tests showed that optimal medium menu included 15g wheat bran,15g yeast extract powder, 2.5g CaCl₂, 0.5g CoCl₂, 0.8g MgSO₄, 0.5g FeSO₄, 0.8g KH₂PO₄ and 1000mL distilled water, pH 7.0. The number of live bacteria could be 3.234×10¹² cfu/mL in the optimized medium and the number increased by one orders of magnitude than original NB medium.