A sensitive spectrophotometric method is developed for determination of content of cationic surfactants (CSs) in water samples. In weak acidic HCl-NaAc buffer medium, some cationic surfactants cetylpyridinium bromide (CPB) and cetyltrimethylammonium bromide (CTAB) react with eosin methylene blue (EMB) to form ion-association complexes, and the maximum absorption wavelength is 660 nm and 658 nm resp. At such maximum absorption wavelength, the color intensity shows good linearity with the concentration of the CS. Within the range 0-2.10 × 10-5 mol/L and 0-1.98 × 10-5 mol/L of CPB and CTAB resp., the relationship between the color intensity and concentration of the CSs follows Beer's Law. The apparent molar absorptivity ε is 1.79 × 104 L/(mol/cm) and 1.38 × 104 L/(mol/cm) resp. and the detection limit is 8.27 × 10-7 mol/L and 9.88 × 10-7 mol/L resp. Average recovery is 99.5-102.6%. The method has high sensitivity and fairly good selectivity. This method can be applied for the determination of cationic surfactants in water samples.