As the critical micelle concentration (CMC) is one of the essential parameters of surfactant, the determination of CMC value is of great significance. We explored a facile and novel strategy based on the visible-light-induced oxidase-like activity of fluorescein for determining the CMC value of cationic surfactants. Fluorescein as a visible-light-induced oxidase mimic can catalyze the change of 3,3′,5,5′-tetramethylbenzidine (TMB) from colorless to blue (oxidation state TMB) under visible light illumination. Nevertheless, it was worth noticed that cationic surfactant could significantly inhibit the catalytic activity of fluorescein with a concentration-dependent mode due to the electrostatic interaction between negatively charged fluorescein and cationic surfactant in aqueous solution. With the increased of surfactants concentrations, the absorbance of the oxidation state TMB was decreased. As the concentration of cationic surfactant reached CMC, the catalytic activity of fluorescence was almost inhibited completely. Therefore, this novel and convenient spectrometric method provides a new idea for the determination of CMC value and also broadens the application of small molecule mimic enzymes.