A high-speed particle image velocimetry based on 90° Mie scattering was proposed to evaluate the status of laminar flow and single-cell flow stability in the flow cell of a flow cytometer. The velocity stability of the particles in the flow cell was used to evaluate the single-cell flow stability of the flow cytometer. Firstly, 90° Mie scattering images of the particle were acquired with a high-speed image sampling system. The 90° Mie scattering selected could avoid the interference of the excitation light source and could remove the background source and improve the image contrast. Then, the grey cluster analysis algorithm based on trapezoid whitenization weight functions was used to classify for all different types of images with shortages, normal, diffraction and overlap. Finally, a center-point method was taken to determine the boundaries of the trailing image to improve the computational accuracy of the length of particle trailing. A experimental system was carried out to verify the feasibility of the comprehensive method. The results show that the method obtains distinct trailing images and they have been classified accurately. Moreover, for the system of this article, the average length of trailing image covers 116.9 pixels, and the standard deviation is 1.7.