Spot size and collimation performance of excitation light are important indexes to evaluate the performance of fluorescence excitation light path of nucleic acid detection system. This paper introduces a calibration method of fluorescence excitation light path performance for nucleic acid detection system. Firstly, the excitation light spot model and optical path collimation model are established, and the numerical calculation is carried out by MATLAB. Then, the accuracy of the model is preliminarily verified by ZEMAX ray tracing simulation. Finally, the excitation light path collimation performance test platform is built, and the industrial cameras are used to collect the light spot for experimental verification. The maximum output spot of the excitation light path is 2.23 mm, and the output angle is 1.72°. The results show that the established spot model and optical path collimation model can be used to calibrate the excitation optical path performance of nucleic acid detection instrument, which can effectively solve the problem that the spot is difficult to be accurately controlled, and provides a new idea for optical path collimation evaluation.