Abstract: A new method for proteinase A activity detection using resonance light scattering technique which was named as RLS method was established. The existing proteinase A activity detection methods and RLS method from the aspects of standard curve, precision, detection limit and the actual sample testing were compared. The result showed, RLS method had lower detection limit (0.662μg/mL) , wider linear range (1⁵00μg/mL) , higher determination coefficient (R2=0.9974) and lower relative standard deviation (RSD=1.95%) than UV method, Lowry method and Bradford method. In addition, this method had obvious advantages in detection of actual samples. Validation test proved that RLS method was accurate and reliable, and the determination coefficient between this method and fluorescent substrate method was 0.9917.