Objective A novel photosensitizer I was synthesized in our laboratory by linking photosensitizer chlorin with folic acid, a targeting group, through PEG linker. The aim of this paper is to evaluate the targeting and photodynamic activities of photosensitizerⅠfor laryngeal carcinoma Hep-2 cells and nasopharyngeal carcinoma CNE cells. Methods The expression of folate receptor in Hep-2 cells and CNE cells was detected by Western blot. The cellular phagocytosis of photosensitizerⅠby Hep-2 cells and CNE cells with or without the presence of excessive folic acid were tested with fluorescence spectroscopy. The cytotoxicity of photosensitizerⅠagainst Hep2 cells and CNE cells with or without irradiation, and the effects of photosensitizer concentration and the irradiation dose on the viability of Hep2 cells and CNE cells were measured by MTT assays. Results Western blot analysis revealed moderate expression of folate receptor in Hep2 cells, and weak expression in CNE cells. The cellular phagocytosis of photosensitizer I by both Hep2 cells and CNE cells increased with the concentration of photosensitizer, and the phagocytic activity of Hep2 cells was stronger than that of CNE cells (P<0.01). The presence of excessive free folic acid could competitively inhibit the phagocytic activity of Hep2 cells. The results of MTT assays demonstrated that photosensitizerⅠexhibited significant photocytotoxicity for both Hep2 cells and CNE cells, and the photodynamic activity of photosensitizer I was positively correlated with the concentration of photosensitizer and the irradiation dose. Conclusion Photosensitizer I displays clear targeting and photodynamic activity for laryngeal carcinoma Hep2 cells with high level expression of folate receptor.