Sturgeon, as a high‐yield economic fish species, is widely consumed, while large quantities of the liver are discarded. Three‐phase partitioning (TPP) was developed in this study as an emerging technology which can well extract oil and protein from sturgeon liver at the same time. Through single‐factor experiments and response surface analysis, we determined that the optimum extraction conditions were as follows: 36.65% mass fraction of ammonium sulfate, 2.88:1 liquid–solid ratio and pH 4.70. The yields of protein and oil were 70.08% and 86.77%, respectively, with an average yield of 78.44%. The qualities of obtained protein and oil were studied, and the results show that the protein and oil prepared by TPP both have high nutritional value and application value. Therefore, TPP would be a better alternative for extracting the oil and protein from sturgeon liver. Practical applications Both protein and lipid can be extracted from sturgeon liver by three‐phase partitioning (TPP). The results show that TPP is a feasible method for simultaneously obtaining protein and oil.