An investigation on the photobleaching behavior of fluorescein in microscopy was carried out through a systematic analysis of photobleaching mechanisms. The individual photochemical reactions of fluorescein were incorporated into a theoretical analysis and mathematical simulation to study the photochemical processes leading to photobleaching of fluorescein in microscopy. The photobleaching behavior of free and bound fluorescein has also been investigated by experimental means. Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. The simulation suggests that the non-single-exponential behavior is caused by the oxygen-independent, proximity-induced triplet-triplet or triplet-ground state dye reactions of bound fluorescein in microscopy. The single-exponential process is a special case of photobleaching behavior when the reactions between the triplet dye and molecular oxygen are dominant.