Abstract: 【Objective】The study was conducted to explore the optimum formula and fermentation condition of liquid fermentation medium for Agaricus blazei Murill.【Methods】With the dry weight of A. blazei mycelium as the index, the liquid fermentation medium formula was optimized by single factor experiments and orthogonal experiments. 【Results】Under the conditions of single factor experiment, the optimal fermentation conditions were taking mannitol as carbon source, (NH4)2SO4 as nitrogen source, NaH2PO4 as other nutrient element with pH of 5.0. Through orthogonal design of different culture combinations, the best formula of A. blazei liquid fermentation medium were taking glucose as carbon source, (NH4)2SO4 as nitrogen source and NaH2PO4 as other nutrient element with the initial pH of 6. After conducting 150 r/min shaking culture at 25 ℃ for 5 days, the dry weight of A. blazei mycelium in 100 mL fermentation broth reached 0.4463±0.0132 g.【Conclusion】The results showed that there was a certain difference between single factor culture and orthogonal combination culture. In order to determine the culture and fermentation conditions more accurately, it is needed to combine the two methods when conducting fermentation culture. In addition, the liquidfermentation of A. blazei can rapidly achieve a large number of strain expansion, which lays a foundation for further cultivation or extraction of fermentation products.