Liquid spawn, an important technical support for industrial production of edible fungi, has some advantages, such as fast growth, strong activity, uniform quality and easy inoculation. However，the study of the fermentation parameters of Pleurotus eryngii liquid spawn is less, which cannot effectively guide the production practice. Therefore, in order to provide a theoretical basis for the production of liquid spawn and industrial production of Pleurotus eryngii, response surface analysis was used to optimize the liquid spawn formula and conditions. The best carbon and nitrogen source of liquid medium was glucose and yeast extract, respectively. The optimum formula of medium was obtained by response surface analysis. Combined with the practical operation, the formula was modified to 29.00 g/L glucose, 2.90g/L yeast extract, 0.90g/L KH2PO4 and 1.00g/L MgSO4. The effects of various factors on the dry weight of mycelia were analyzed, the results indicated that the influence follow the sequence: glucose, KH2PO4, yeast extract and MgSO4, among them, the first two factors had significant effect. The interaction between glucose and KH2PO4 had obviously effects on the dry weight of mycelia. The interaction between glucose and yeast extract had a certain effect. The optimum culture conditions of liquid spawn were obtained by response surface analysis. Combined with the actual situation, the conditions were modified to liquid volume 106.00mL/250mL, rotating speed 165.00r/min, temperature 23.60℃, initial pH 6.70. The effects of various factors on the dry weight of mycelia was analyzed, the results indicated that the influence follow the sequence: initial pH, liquid volume, temperature and rotating speed, among them, the first three factors had extremely significant effect. The interaction of liquid volume and rotating speed, temperature and initial pH had obviously effects on mycelial dry weight.