The invention discloses a culture method of a high-protein pleurotus eryngii liquid strain. The culture method comprises the following steps of preparing materials, processing potatoes, taking juice, mixing potassium dihydrogen phosphate and magnesium sulfate, adding the potato juice, fully mixing, adding white sugar, soybean meal, vitamin B, vegetable oil and peptone, adding water, and mixing in stirring equipment, sterilization treatment: putting the blended mixed juice into a culture tank to prepare a culture solution, and inoculating culture: placing the mother strain in sterile water, when a culture solution is cooled to 30 DEG C, opening a tank for inoculation, then sealing a cover immediately, introducing sterile air for culture, acquiring a liquid strain, and injecting the liquid strain into a pleurotus eryngii cultivation bag after being inspected to be qualified. According to the culture method of the high-protein pleurotus eryngii liquid strain, the influence of bacteria in the fermentation process can be reduced, and the strain quality is improved; the pollution of harmful gas generated by fermentation to the environment can be reduced, and the environmental protection property of the culture process is improved.