Objective  To optimize the determination method of Staphylococcus aureus α-toxin (AT) hemolytic activity for in vitro functional evaluation and neutralizing antibody titer determination of non-toxic mutant α-toxin (mAT) specific serum antibody.  Methods   Rabbit red blood cells were lysed with AT to release hemoglobin. The hemolysis activity was detected by spectrophotometry. The incubation time, rabbit red blood cell final concentration, and Triton X-100 concentration of the method were optimized, and the repeatability of the method was verified. In vitro functional evaluation of mAT-specific serum antibody was carried out, AT hemolysis curve was drawn, the concentration of AT with 50% hemolysis rate was calculated, and the serum 50% neutralization titer (NT₅₀) was detected.  Results   The optimal conditions for detection of AT hemolytic activity were incubation time 90 min, rabbit red blood cells final concentration 2% (V/V), and Triton X-100 concentration 0.25%. Under these conditions, respectively adding serum antibody of mAT and multi-antigen containing mAT both significantly inhibited the hemolytic activity of AT. When the hemolysis rate was 50%, the AT concentration was 1.75 μg/ ml and coefficient of varriation was 3.75%. The preliminary results of NT₅₀ of mAT and multi-antigen serum antibody containing mAT were both 1∶64.  Conclusion  The optimized AT hemolytic activity assay has good repeatability and short detection time, which can be used for functional evaluation of specific serum antibodies and titer determination of neutralizing antibodies in vitro.