Abstract: The accurate quantification of proteins in extracellular polymeric substances (EPS) is of great significance for studying the properties of activated sludge. To provide a basis for the selection of protein quantification methods in EPS, the test performance of Modified Lowry method, BCA method, Bradford method was systematically evaluated, including the test range, accuracy, precision and the anti-interference of some components (Ca²⁺, Mg²⁺,reducing sugar and humic acid) in EPS. The results indicated that the Modified Lowry method showed a good linear correlation between protein concentration and absorbance in the range of protein concentration of 1-10 and 10-100 mg/L (R²>0.99); and the test range of BCA method and Bradford method was wider. Modified Lowry method had the highest accuracy and precision, BCA method ranked the second, and Bradford method was the worst. Besides, Modified Lowry method was affected by Ca²⁺, Mg²⁺and reducing sugar; BCA method was affected by reducing sugar and humic acid; and Bradford method was affected by humic acid. Therefore, the choice of protein quantification method in EPS should be comprehensively considered from the two aspects of sample properties (protein content range, interference species and concentration) and test method performance (test range, accuracy, precision and anti-interference).