The fluorescence microscopy imaging system is one of the key modules of the gene sequencer. In order to ensure the accuracy of gene sequencing， the fluorescence imaging system must be able to achieve high resolution and high-quality imaging. To meet the requirements of miniaturization， low cost， and high resolution for gene sequencers， this paper designed a dual band gene sequencing fluorescence microscopy imaging system based on narrowband LED illumination. Firstly， based on the basic principle of fluorescence microscopy imaging， an LED based illumination system was designed using the critical illumination principle. The illumination uniformity was optimized through simulation analysis， and a high-resolution and high imaging quality imaging system was designed using a beam splitter. The prototype development was completed through the design scheme， and the imaging performance was verified. According to the test results， the designed microscopy system has a resolution of 0.8 μm in the XY direction. The illumination uniformity is better than 80%， meeting the requirements of high-resolution imaging. Compared with other systems， this system has the characteristics of miniaturization and low cost， meeting the needs of gene sequencing， and has important application prospects in the development of miniaturized gene sequencers.