Precisely distinguishing between hyperplastic and adenomatous polyps and normal human colonic mucosa at the cellular level is of great medical significance. In this work, multiphoton laser scanning microscopy (MPLSM) was used to obtain the high-contrast images and the morphological characteristics from normal colonic mucosa, hyperplastic polyps and tubular adenoma. By integrating the length and area measurement tools and computing tool, we quantified the difference of crypt morphology and the alteration of nuclei in normal and diseased human colonic mucosa. Our results demonstrated that the morphology of crypts had an obvious tendency to cystic dilatation or elongated in hyperplastic polyps and tubular adenoma. The content and number of mucin droplets of the scattered goblet cells had a piecemeal reduction in hyperplastic polyps and a large decrease in tubular adenoma. The nuclei of epithelial cells might be elongated and pseudostratified, but overt dysplasia was absent in hyperplastic polyps. Nevertheless, the nuclei showed enlarged, crowded, stratified and a rod-like structure, with loss of polarity in tubular adenoma. These results suggest that MPLSM has the capacity to distinguish between hyperplastic and adenomatous polyps and normal human colonic mucosa at the cellular level.