Abstract: In order to obtain a low-cost fermentation medium for Pediococcus acidilactici PA003 in a large scale, the fermentation medium and fermentation conditions were optimized by single factor test and orthogonal test. The results showed that the best compositions of fermentation medium were 2% of molasses, 2% of soybean powder, 0.3% of corn extraction solution, 0.2% of dipotassium hydrogen phosphate, 0.5% of sodium acetate, 0.2% of ammonium citrate, 0.058% of magnesium sulfate, 0.025% of manganese sulfate, and 0.1% of Tween 80, and the pH of the medium was 6.5. The results also indicated that the best fermentation inoculum size was 6%, incubation temperature at 37℃ and time for 14h, speed at 140r/min. The biomass of Pediococcus acidilactici in such conditions reached 6.4×109CFU/mL, which was equivalent to the biomass in MRS medium.The results of this study suggested good application prospect in industry.