A mass of reactive oxygen species(ROS) are produced in the process of smoking. Superfluous ROS can induce the oxidative stress in organism, which will cause irreversible damage to cells. Fluorescent probe is taken as a marker of oxidative stress in biology and has been applied to ROS detection in the field of biology and chemistry for high sensitivity, high simplicity of data collection and high resolution. As one type of fluorescent probe, no-fluorescent dihydrorhodamine 6G(dR6G) will be oxidized to the fluorescent rhodamine 6G. In this process, each molecule reacts with two active oxygen molecules. The probe can be used to detect ROS in mainstream cigarette smoke by using derivative fluorescence method. The action mechanism of ROS on dR6G was investigated and the standard curve of R6G fluorescence intensity with its content was built up. The contents of ROS from the 4 types of cigarettes purchased in market were detected and the cleaning ability of filter tip to ROS in cigarette smoke was also researched. The result shows that the amount of ROS has close relationship with the types of tobacco and cigarette production technology and the cleaning ability of filter tip to ROS in cigarette smoke is very limited. Compared with other detecting methods such as electronic spin resonance(ESR), chromatography and mass spectrometry, this detection method by the fluorescent probe has higher efficiency and sensitivity and will have wide applications in the ROS detection fields.