The invention discloses a method for utilizing a two-dimensional electrophoresis system for obtaining a two-dimensional electrophoresis graph of tilapia muscle protein. The method comprises the following steps that soluble tilapia protein used for two-dimensional electrophoresis is prepared; concentration of the soluble tilapia protein used for the two-dimensional electrophoresis is detected; the two-dimensional electrophoresis is prepared and comprises isoelectric focusing electrophoresis, adhesive tape balance and sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis; gel dyeing is carried out, wherein a silver nitrate dyeing method is adopted for dyeing SDS-PAGE gel; graph scanning is carried out, wherein a scanner is used for scanning the gel to obtain the two-dimensional electrophoresis graph of the tilapia muscle protein. The method is easy to operate and suitable for the two-dimensional electrophoresis system of the tilapia muscle protein so as to obtain the two-dimensional electrophoresis graph of the tilapia muscle protein, the foundation is laid for follow-up study of the tilapia muscle proteomics, and the reference is provided for study of muscle proteomics of other fishes.