Abstract: Based on the electrostatic reaction between anionic surfactants and the cation of methylene blue to form a blue compound which can be extracted with CHCl3 and to have its absorbance detected at the wavelength of 652 nm. Spectrophotometry was applied to the determination of anionic surfactants in water. The optimized conditions found were as follows: ① amount of 0.09 g·L-1 alkaline methylene blue solution added: 25 mL; ② condition of extraction: extract for 3 times with 45 s in each extraction. Interferences of protein and quaternary ammonium compounds were eliminated by the alkalinity of the methylene blue solution. Linear relationship between values of absorbance and mass concentration of anionic surfactants was obtained in the range of 0.100-2.00 mg·L-1, with detection limit (3s) of 0.005 mg·L-1. Values of recovery found by standard addition method were in the range of 91.0%-99.3%, and values of RSD′s (n=6) less than 5.0%.