We use a new procedure for estimating acid ionization constants of fluorescein using the spectrophotometric method at 25 degrees C and at the constant ionic strength I = 0.1M (KNO3) in pure water as well as in aqueous media containing variable percentages (10-50% w/v) of organic solvents. The used organic solvents were methanol, ethanol, N,N-dimethyl formamide, dimethyl sulfoxide, acetonitrile, and dioxane. The obtained results indicated that acidity constants decrease as the content of an organic solvent in the medium increases. The acidity constants of all related equilibria are estimated using the whole spectral fitting of the collected data. The DATAN program was applied for determination of acidity constants. The effects of various solvents on absorption properties and acidity constants of each component are also discussed.