The hyperspectral SPRi sensor was used to perform microscopic analysis of polystyrene microspheres, and the hyperspectral SPR data collected under different polarization conditions were processed, resulting in effective suppression of the contrast of inherent stains in the SPR hyperspectral images and making the position and outline of the polystyrene microsphere sample clearer. The processed SPR spectra become smooth, reducing the influence of the spectral characteristics of the light source and random noise on the SPR resonance information extraction. By comparing the SPR image of polystyrene microspheres with the reflection bright field microscopic image, it is found that there are obvious differences between the two images. The main reason is the limitation of the penetration depth of the evanescent field. The results show that SPR images can clearly reflect the contact between particles and metal surfaces in the evanescent field, but cannot quantify the true size of objects beyond the penetration depth of the evanescent field compared with reflection bright field microscopy images.