The reaction between fullerenol (F-ol) and cytochrome C (Cyt C) could be carried out to form a nonphosphorescence compound using tween-80 as photosensitizer which causes the sharp quenching of room temperature phosphorescence (RTP) of F-ol. Bearing this in mind, a novel solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace Cyt C has been proposed in this study. Under the optimum conditions, the linear range of this method is 1.0×10-16 –2.4×10-14 g mL-1, which is directly proportional to ΔIp of Cyt C-F-ol–tween-80 system, and the detection limit (DL) is 3.0×10−17 g mL-1. It has been applied to the determination of Cyt C in human serum and forecast of diseases, and the result matches with the enzyme-linked fluorescence immunoassay (ELISA). Meanwhile, the reaction mechanism of SSRTP for the determination of Cyt C and the enhancing effect of tween-80 on RTP of F-ol were also discussed.