Single molecule immune detection refers to limiting immune complexes to a very small volume and counting the generated signals. It is a "digital" immune detection technology. The core of the single molecule immune detector is similar to a fluorescence microscope system, but the common fluorescence microscope has a complex structure and cannot match the specification of the biochip, which cannot meet the detection requirements well. A set of high performance optical system for single molecule immune detection is summarized according to the specification of biochip developed by ourselves. Firstly, the appropriate initial structure is determined according to the design requirements; Secondly, on the basis of fluorescence microscope system principle and aberration analysis theory, the optical design software Zemax is used to repeatedly optimize the system design; Finally, an optical system is designed to meet the imaging requirements of single molecule immune detection. The imaging part of the system consists of 15 pieces of refractive lenses, the working object distance is 4mm, the magnification is -4.52, the modulation transfer function values are greater than 0.44 at the Nyquist frequency of 73lp/mm, and the distortion is 0.8%, Kohler lighting mode is adopted for the lighting part, and the illuminance uniformity on the object surface reaches 97% in the whole field of view. The whole system adopts domestic conventional glass, which is conducive to processing and cost reduction. The optimized high resolution fluorescence microscopy system combined with the current fully automated detection technology can greatly improve the detection sensitivity and accuracy of single molecule immunoassay.