Abstract: Chrome azure S (CAS) and Cu ions form blue-purple complex under the existence of hexadecyl trimethyl ammonium bromide. EDTA and methanobactin(mb) may change the color of the CAS-Cu complex, respectively, because of the copper-binding ability. In the spectrophotometric method, the copper-binding curve of EDTA and methanobactin can be drawn, and the relative concentration of mb to EDTA can be calculated by a regression equation. Now a spectrophotometric method to detect methanobactin is designed, the maximum absorbance is 605 nm,and linear regression equation is Y=-3.5X+0.00536，and R²=0.9946, showing the linear relationship is good. The test results of practical samples show that this method is simple, sensitive, accurate and reproducible.