An electrically controlled optical chopper based on holographic polymer dispersed liquid crystal grating (H-PDLC) and its application within the fluorescence microscopy has been investigated in this paper. The H-PDLC chopper has the characteristics such as free waveform, easy and precise tunable working frequency, no moving parts etc. In order to testify its application, a two-channel frequency division multiplexing confocal fluorescence microscopy (FDMCF) has been established, where the integrated H-PDLC chopper array modulates the individual exciting laser beam with different carrying frequency. The FDMCF microscopy detects two-channel fluorescent signals from the two different spots of the specimen’s surface synchronously. The successful experiments testified the optical chopper device based on H-PDLC technique, and explored its application possibility within the modern integrated optical system as well.