Flow cytometers are instruments used for the rapid quantitative analysis of cell suspension. Traditional flow cytometry uses multi-channel filters to detect fluorescence, whereas full-spectrum fluorescence based on dispersion detection is a more effective and accurate method. The application of various dispersion schemes in flow cytometry spectroscopy has been studied. From the perspective of modern detectors and demand for the miniaturization of flow cytometry, prism dispersion exhibits higher and more uniform light energy utilization, meaning that it is a more suitable dispersion method for small flow cytometers, such as microfluidic flow cytometers. Prism dispersion designs include the size, number, and placement of prisms. By deducing the formula of the final position of light passing through the prism and combining it with the formula of transmittance, the design criteria of the top angle and the incident angle of the prism in pursuit of the optimum transmittance and dispersion index can be obtained. Considering the case of multiple prisms, under the premise of pursuing a smaller size, the optimal design criteria for dispersion system composed of multiple prisms can be obtained. The design of prism dispersion fluorescence detection was demonstrated with a microfluidic flow cytometer, and the effectiveness of the design results was verified by microsphere experiments and practical biological experiments. This design criterion developed in this study is generally applicable to spectral flow cytometers.