The invention discloses a quick sperm acrosin extraction method and a sperm acrosin activity flow cytometry detection method. The extraction method is characterized by including: adding seminal fluid into a centrifuge tube, centrifuging to remove seminal plasma, keeping lower sperm precipitates, adding membrane rupture liquid to enable uniform suspension of sperm precipitates, centrifuging to remove supernatant, adding extracting solution to make sperms suspended, and centrifuging to collect supernatant, so that acrosin extract is obtained. The sperm acrosin activity flow cytometry detection method is characterized in that by means of polypeptide or protein microsphere coupling and polypeptide or protein fluorescence labeling, an acrosin digested substrate is subjected to fluorescence labeling and then connected to the microspheres, fluorescent microspheres in certain particle diameters can be recognized by a flow cytometry instrument, and fluorescence intensity directly reflects activity level of acrosin in a sample. By combination of quick extraction and flow cytometry, detection efficiency is greatly improved, and detection sensitivity is improved.