A novel method for direct determination of hemolysin activity in antiserum by linear–sweep polarography(LSP) was developed in this paper. This method is based on a reduction wave of active hemolysin in phosphate buffer solution. The peak height of hemolysin is linear proportional to its activity in the range of 6×10−6unit/mL to 2×10−3unit/mL. Deactivated hemolysin, other proteins and chemicals present in biological fluids do not interfere in the assay. This method is very rapid, simple, highly specific and sensitive.