Measurement of dissolved oxygen (DO) in liquid samples is of vital importance in both industrial and biomedical fields. In this paper, a DO sensor based on the fluorescence quenching method has been built. The measurement principle is based on fluorescence lifetime detection, which is indicated by the phase difference between an excitation light signal and a fluorescence signal. The nonlinear effect of the fluorescent material has been taken into consideration to obtain a more accurate fitting model. The performance of the system varying with the modulation frequency of excitation light signals is also reported. Modulation frequency mainly affects the sensitivity and phase resolution ratio of the system. The system at the optimized modulation frequency has a good degree of fitting with R2 value of 0.9981 and a small relative error of 0.79%. The study shows that this kind of sensor with optimal modulation frequency has good performance, which can be used in many important fields.