IFN-gamma and TNF-alpha were co-coupled to the polystyrene cell culture plate by the photo-immobilization method. To investigate the synergistic effect of IFN-gamma and TNF-alpha on the HeLa cells, HeLa cells were treated with co-coupled cytokine or non-coupled cytokine in a time course in this study. The morphology detection, cell cycle analysis by flow cytometry, phosphatidyl serine analysis and capase-3 activity detection demonstrated that the two kinds of treatments both induce HeLa cells apoptosis. Non-coupled cytokine worked more quickly while co-coupled cytokine kept more permanent effect. The caspase-3 activity assay indicated that the caspase-3 activity of HeLa cells treated with non-coupled cytokine is higher than that of HeLa cells treated with co-coupled cytokine. This may imply that co-coupled cytokine not only induces the caspase-dependent pathway, but also induces the caspase-independent pathway.