Abstract: The experiment used 5, 5'-dithianitrobenzoic acid (DTNB) colorimetric method to quantify metallothioneins (MTs) content, pretreatment of samples by acid hydrolysis. The optimal condition was determined by single factor and orthogonal test:24 hours hydrolysis, 6 mol/L hydrochloric acid, 1:20 mass volume ratio, and hydrolysis temperature at 130℃. The optimized hydrolysis degree could achieve 55.28%, with detection of MTs 0.4821 mg/g. After pretreatment, the reaction conditions and reagent preparation conditions were optimized. Ellman reagent was preparal with disodium hydrogen phosphate-citric acid buffer with pH4.0, the best reaction pH was 7.2. The experiment showed a good linear regression (Y=0.019x+0.027, R²=0.999). Besides, the recovery test result of its standard was 75.83%~88.15%, and the relative standard deviation was 3.10%. The method had relatively good reproducibility, accuracy and precision with detection limit 7.57 μg/g. This method could be used for rapid detection of MTs.