Mixed culture fermentation were used in this experiment to investigate new methods for improving the quality of nutrition of Pleurotus eryngii residue as a feed. A L9(34) orthogonal design was used for this experiment: Trichoderma longicornis(5×103, 5×104, 5×105 cfu/g), Bacillus subtilis(5×105, 5×106, 5×107 cfu/g), Saccharomyces cerevisiae(5×106, 5×107, 5×108 cfu/g), and Pichia jadinii(5×105, 5×106, 5×107 cfu/g) were inoculated into the Pleurotus eryngii residue, recorded as a, b, c, d, e, f, g, h, and i, respectively; and a control group(without microorganisms). The indicators of nutrients in the Pleurotus eryngii residue were detected after 144 hours’ continuous fermentation in an anaerobic environment; then followed by a simulated rumen fermentation in vitro for 48 hours before the indexes collected. The result of the experiments had shown that, compared to the control group, the mixed culture fermentation increased significantly the crude protein mass fractions of the a, c, d, f, g, h, i treatment groups and the crude fat mass fractions of the a, b, c, d, f, h treatment groups; and reduced significantly the neutral detergent fiber mass fractions of the d treatment group and the acid detergent fiber mass fractions of the a, d treatment groups. The four microorganisms all played roles to affect the effects in the process, Bacillus subtilis, Saccharomyces cerevisiae and Pichia jadinii played significant roles especially. The mixed culture fermentation did not affect significantly the rumen fermentation of Pleurotus eryngii residue. According to the analysis, the optimum treatment was that the inoculation levels of Trichoderma longicornis, Bacillus subtilis, Saccharomyces cerevisiae and Pichia jadinii were 5×104, 5×105, 5×107, 5×107 cfu/g, respectively.