The invention discloses a method for extracting and detecting fish sperm membrane protein. The method comprises the following steps: pretreatment of a sample, fabrication of sperm suspension, suspension treatment of sperms, sperm membrane protein separation, sperm membrane protein degreasing purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection of sperm membrane protein and mass spectrometric detection of the sperm membrane protein. By adopting the method, the characteristics of a protease inhibitor and a TM-PEK (transmembrane-protein extraction kit) method are fitted on the basis of the traditional decontamination method, the cover degree of the extracted fish sperm membrane protein can be up to over 86%, and 0.6mg of membrane protein and 0.8mg of cytoplasmic protein can be obtained from 5*10<7> sperms on average. The basic data can be provided for verification of a fish sperm egg mechanism and a fertilization mechanism by the popularization and application of the method disclosed by the invention, guidance can be provided for culture and proliferation of fishes, especially reproductive breeding of migratory fishes in estuary sea, and a useful reference method is provided for research of sperm membrane protein of other aquatic species.