A fluorescence microscope detection system based on digital micromirror device (DMD) is designed. The system not only can realize the fluorescence imaging of the sample, but also can detect the real-time change of the fluorescence signal energy in different regions of the sample. The video information with different frame rates in multi-channel is fast loaded by DMD in the experiment to realize the segmentation of the excitation beam and frequency modulation. The fluorescence information of the sample is collected by photomultiplier tube (PMT) and then is processed by means of analog-to-digital conversion, Fourier transform, filtering and demodulation, and the relationship curve between intensity of fluorescence signal in different channel regions and time. The experimental results show that adding DMD to fluorescence microscope system can accelerate the integration of multi-channel, the DMD channel can be controlled by computer, and the detection efficiency of the fluorescence microscope can be improved effectively.