The microenvironment in reverse micelles composed of water, aerosol OT (AOT, sodium bis(2-ethylhexyl)sulfosuccinate), and isooctane has an important effect on the spectral properties of fluorescein (FL). The absorption peaks of FL red-shifted by about 10 nm from the corresponding positions in aqueous solution. Moreover, the absorption extinction coefficient of FL in reversed micelles increased with the water content, omega. The fluorescence of FL was more effectively quenched in AOT reversed micelles than in aqueous solution. In addition, aggregation occurred for FL in reversed micelles, which hindered the photo-induced electron transfer from the excited singlet of FL to the conduction band of TiO2.