The fluorescence lifetime (τ f ), emission quantum yield (Φ f ), absorption and emission spectral data of 20 fluorescein derivatives were measured under the same conditions by using time-correlated single photon counting, steady state fluorescence and absorption methods to get comparable data. Based on the results, the factors and mechanism that control the fluorescence properties of the fluorescein dyes are discussed. Both Φ f and τ f are remarkably dependent on the substitution on either xanthene or phenyl rings, but their ratio (Φ f /τ f ), i.e. rate constant of radiation process, is a constant value (0.20 × 10 9  s −1 ). The rate constant of nonradiation process, on the other hand, is varied with both the structure and the solvent used.