By using Plackett–Burman combined with Box–Behnken design, the fermentation conditions of Pichia burtonii using Agaricus bisporus industrial wastewater as culture medium were optimized. The biomass of P. burtonii in the fermentation broth was analyzed by multispectral imaging flow cytometry. Plackett–Burman design was used to screen out three factors from six factors affecting the biomass of P. burtonii as major factors. The Box–Behnken response surface method was used to optimize the interaction of the three main factors to predict the optimal fermentation conditions. The significant factors affecting the biomass of P. burtonii, such as shaking speed, solubility and culture temperature, were screened. The optimum conditions for P. burtonii were as follows: a shaking speed of 265 rmp, a solubility of 8%, a culture temperature of 25 °C, an initial pH of 6.0, an inoculation amount of 8%, and an amount of 30 mL liquid in 250 mL, and the total living yeast can reach 1.27 ± 0.02 × 108 Obj/mL, which was within the 95% confidence interval of the predicted model (1.08–1.32 × 108 Obj/mL).