Cutaneous squamous cell carcinoma (cSCC) is the second most common malignant skin tumor. Early and precise diagnosis of tumor staging is crucial for long-term outcomes. While pathological diagnosis has traditionally served as the gold standard, the assessment of differentiation levels heavily depends on subjective judgments. Therefore, how to improve the diagnosis accuracy and objectivity of pathologists has become an urgent problem to be solved. We used multispectral imaging (MSI) to enhance tumor classification. The hematoxylin and eosin (H&E) stained cSCC slides were from Shanghai Ruijin Hospital. Scale-invariant feature transform was applied to multispectral images for image stitching, while the adaptive threshold segmentation method and random forest segmentation method were used for image segmentation, respectively. Synthetic pseudo-color images effectively highlight tissue differences. Quantitative analysis confirms significant variation in the nuclear area between normal and cSCC tissues ( < 0.001), supported by an AUC of 1 in ROC analysis. The AUC within cSCC tissues is 0.57. Further study shows higher nuclear atypia in poorly differentiated cSCC tissues compared to well-differentiated cSCC ( < 0.001), also with an AUC of 1. Lastly, well differentiated cSCC tissues show more and larger keratin pearls. These results have shown that combined MSI with imaging processing techniques will improve H&E stained human cSCC diagnosis accuracy, and it will be well utilized to distinguish histopathological staging features.