Fluorescence immunochromatographic quantitative detection technology has been widely used in the field of clinical detection, such as for the screening and detection of the new coronavirus pneumonia. To improve the detection accuracy, a fluorescence immunochromatographic quantitative detection method based on fluorescence microscope digital image processing is proposed. Initially, the luminous flux loss equation of the microscopy system is constructed. Then, the RGB components in the original image are extracted according to the digital image features output by the area array CCD, and the quantum response rate curve of the area array CCD is fitted after weighting to compensate. Finally, the relative intensity of the fluorescence signal is obtained, and the concentration of the analyte is inversed according to the intensity of the fluorescence signal. After experimental verification, it can be observed that the coefficient of variation of the test results is 3.04%, and the linear fitting coefficient is >0.99. This detection method can accurately detect the analyte with a minimum mass concentration of 0.1 ng/mL. It is suitable for CCD imaging fluorescent microscope system and has certain reference significance for fluorescence detection.