Ocular surface squamous neoplasia(OSSN)is the non-pigmented ocular surface malignancy presented as prominent vascularity, severe irritation and vision loss. Topical chemotherapy with IFNα2b and 5-FU has been widely recognized as effective supplements. The cooperative effect of IFNα2b and 5-FU on OSSN is theoretically based on the regulation of IFNα2b on key enzymes in DNA damage mediated by 5-FU. This study explores the underlying effects and related combination mechanisms of IFNα2b and 5-FU on proliferation, migration, apoptosis, and cell cycle of human oral squamous carcinoma Cal27 cell line. Cal27 cell lines were treated with different concentrations of IFNα2b and 5-FU alone or in combination for 48 hours. Then cell activity, tumor cell migration, the combination index (CI value), apoptosis and the expression of key enzymes TP, TS and DPD of 5-FU were detected. These results suggest both IFNα2b and 5-FU alone and in combination can inhibit the proliferation and migration of Cal27 tumor cells, induce cell apoptosis, and arrest cell cycle. Low doses IFNα2b increase the antitumor effect of 5-FU on Cal27 potentially through up-regulating the expression of TP.