According to the needs of the detection of fluorescence with low concentration and short lifetime in the OH radical measurement based on fluorescence assay by gas expansion, a gated photon counting technique for resonance fluorescence detection was described, which was actualized by designing a nanosecond gating circuit applied to photomultiplier. Fluorescence was detected by a high gain 13-stage end-window photomultiplier, whose gating was actualized by changing the voltage of the photomultiplier dynodes. The circuit could obtain stable modulation voltage, with a 168 ns delay time and a 20 ns rising time. After optimizing the components and matching parameters of gating circuit, the radio frequency noise introduced by the circuit could be reduced in 210 ns and the on/off extinction could be up to 105 by switching the voltage applied to the first, third and fifth dynode of the photomultiplier. The gating system was applied to the fluorescence assay by gas expansion system for fluorescence detection and the OH radical fluorescence signal was obtained. The excitation spectrum of OH radical was measured in the wavelength range of 307.8~308.2 nm. Experiments show that the proposed gating technique can be used to suppress the influence of laser stray light, and detect the OH radical fluorescence signal in a particular time sequence.