On-site rapid detection of nucleic acid has the advantages of convenience, simple operation, no need for special laboratories, and rapid reporting, which can be detected in various complex environments such as airports, community hospitals, customs and fields. A fluorescence detection system that can be applied to on-site rapid detection instruments of nucleic acid was established, including non-confocal orthogonal optical path and multi-channel detection integrated structure. The former has the characteristics of high signal-to-noise ratio, small and convenient. The latter has high switching efficiency and can meet multiple detection, and its embedded structure greatly reduces the fluorescence crosstalk between channels. The experimental results show that the detection limit of the system is lower than 1.6 μg/mL, the R² value (coefficient of determination) of fluorescence detection gradient is greater than 0.99, and the coefficient of variance (CV) value of repeatability test does not exceed 1.27%. The inter-channel fluorescence crosstalk test shows that there is no crosstalk between channels, and the accuracy and stability of cytomegalovirus culture medium amplification experiment are good, which indicates that the system can effectively stimulate and collect fluorescence for multiple detection.