Optical fluorescence microscopy has gradually become a key device in the field of biomedicine, due to its low phototoxicity and easy 3D imaging. This system is a large-value aperture microscope objective designed by ZEMAX, which is characterized by imaging optimization for commonly used fluorescence wavelengths (460nm/509nm/590nm/620nm), and introduces a diaphragm structure to make the focal depth of the objective adjustable, thereby improving the adaptability to the thickness of the optical chip, reducing stray light interference, and increasing the signal-to-noise ratio of the system. The microlens array is designed at the end of the system, which refers to the pixel size adjustment of the CMOS sensor, which improves the energy utilization of the system. The total length of the objective is less than 45mm, the numerical aperture is 0.7, the magnification is 40x, and the working distance is 0.4mm, all of which meet the national standards.