Reactive oxygen species (ROS) can damage molecules such as lipids and proteins, thus cause oxidative damages to cells. During photosensitization process such as photodynamic therapy (PDT), a large amount of ROS is produced. By monitoring the ROS production, it is possible to evaluate the oxidation caused by ROS and the subsequent biological outcome by a PDT treatment. Results indicate that, there is a long-life delayed chemiluminescence (CL) after the human serum oxidized with photosensitization. This property may be used to monitor CL after turning off the excitation light source, thus achieve a high signal/noise ratio in detection. With the delayed CL detection method, CL was evaluated with various photosensitizes. The technique was also tested in vitro, it was found that the CL signal was readily detectable and showed a linear relationship with the dose of PDT treatment. In conclusion, the detection of real-time CL is tested both in solutions doped with various photosensitizes and in vitro and the cumulated CL can be used as a marker for evaluating the damage to biologic molecule and cell. This method can be used on photosensitization such as photodynamic therapy to monitor the dose.