As the intrinsic parameter of fluorescence signal, the accurate measurement of fluorescence lifetime is very important to fluorescence detecting and analyzing technique. According to the characteristics of fluorescence signal and definition of fluorescence lifetime, principle of phase-locking detection technique of fluorescence lifetime is introduced. Based on phase locked loop circuit, fluorescence lifetime detecting system is designed, which is used to dissolved oxygen detecting with fluorescence method. The experimental results showed that the relative error of measured values and actual values is less than 1.5%, and the response time is less than 60 s.