The autofluorescence of 5-Hydroxytryptamine (5-HT) loaded rat mucosal mast cells (RBL-2H3 cells) was imaged with multi-photon excitation laser scanning microscope (MPELSM). Multi-photon excitated 5-HT relative visible fluorescence is observed in live cells for the first time, and the generating mechanism of 5-HT relative visible fluorescence is preliminarily studied. The spatial distribution of 5-HT in live cells was imaged at high spatial resolution in this present, which provides a new way to study the correlation between 5-HT spatial distribution and content, and the cellular functional state in live tissue or cells.