The method of the extraction and purification of the human skeletal muscle aα-actinin is developed from the method of preparation of α-actinin from rabbit skeletal muscle and chicken gizzard. Myosin was eliminated by Hasselbach-Shneider solution first and then the boric-borate buffer and magnesium chloride were used for depletion to remove the actin and other proteins. After high speed centrifugation, the crude extraction was precipitated by 30% and 35% saturation of ammonium sulfate. The purified α-actinin was obtained by DE-52 ion-exchange chromatography finally. The rabbit anti-human skeletal rnuscleα-actinin antiserum was raised with purified human skeletod muscle α-actinin. The titer of the antiserum identified by double diffusion method was 1:32 and by ELISA methal was about 100000. The high specificity antiserum obtained from each rabbit was about 40 mL.