The method of extracting protein from corn steep liquor by isoelectric point method was studied. The supernatant after extracting crude protein was used to produce single cell protein. Single factor experiment was used to analyze the influence of pH, temperature and time on protein extraction. Response surface methodology was used to determine the optimal process of isoelectric point extraction of protein. The conditions for the production of single cell protein from the supernatant of Saccharomyces cerevisiae and Candida utilis were studied. The results showed that the optimum conditions for crude protein extraction by isoelectric point method were as follows: pH 7.6, extraction temperature 26.4 ℃ and extraction time 20 min. The extraction rate was 77.5%. The conditions for producing single cell protein from the supernatant after extracting crude protein were as follows: Candida utilis was better than Saccharomyces cerevisiae. When the dry matter concentration in the supernatant was 8% and pH was 5.5, the colony number obtained by Candida utilis fermentation reached 1.6×109 CFU/mL.