The invention provides a quantitative detection method of sperm surface Izumo1 protein. The method comprises the following steps: performing separation and pretreatment of a sperm, performing preliminary treatment of the sperm to expose the Izumo1 protein epitope of the sperm, marking the Izumo1 protein of the sperm by using fluorescein, and performing detection by using a flow cytometer. According to Tthe method provided by the invention, utilizes the flow cytometry is utilized to perform quantitative detection on the Izumo1 protein in the human sperm for the first time, directly realizes precise quantification of the Izumo1 protein is directly realized, is easy to realize standardization in operation is easy to realize, is quick and accurate effects are realized, and is high inthe precision is high. Compared with traditional methods for qualitative or semi-quantitative detection of Izumo1 protein, such as cell immunofluorescence and fluorescence in situ hybridization, the method provided by the invention has the advantages of convenient detection, high precision, high speed and the like; the method can be applied to quantitative detection of expression of sperm surface Izumo1 protein and is easy to popularize; and the detection result can be used for performing auxiliary evaluation on the fertilization ability of the sperm, so that the method is suitable for practical application.