The 3,3′, 4′,7 tetrahydroxiflavone (fisetin) is a natural therapeutically active and fluorescent polyhydroxyflavone, with important spectroscopic and biological behavior. Fisetin shows dual emission, with a normal band (N) from the S1→S0 transition and the one generated in the excited state (phototautomer; PT) from the intramolecular proton transfer (ESIPT) process. The influence of different interfaces on the ESIPT process of fisetin was investigated in reverse micelles media (RMs) made of the anionic sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (AOT) and cationic benzyl n-hexadecyl dimethylammonium chloride (BHDC) surfactants, in benzene. The studies were carried out by absorption, emission spectroscopy, steady-state anisotropy and time-resolved fluorescence measurements. Fisetin behavior was also investigated in homogeneous media with special emphasis in water and benzene, which are the polar core and the organic pseudofase in the RMs, respectively. In addition, the effect of concentration in benzene and the variation of the pH in water were studied. Fluorescence lifetime measurements show that in water the ESIPT process is independent on the concentration, while in benzene it was possible to detect fluorescent aggregate species (Nas) formed in the ground state. The effect of the pH in water allowed us to identify the anionic fisetin (A−) emission. The studies in RMs show that fisetin interacts specifically with the head of the surfactants, which always results in diminishing the emission of the PT. Also the formation of A− is detected particularly at W0 > 0. Appreciable high anisotropy values are obtained in RMs, as compared with those in fluid homogeneous media, which are independent of the water content confirming that fisetin molecules are anchored in the anionic as well as in the cationic interfaces.