Objective：To investigate and establish the microscopic features of colorectal diseases via multiphoton microscopy（multiphoton microscopic imaging，MPM）initially，evaluating the diagnostic value in vitro. Methods：The multiphoton images of 100 fresh tissues confirmed pathologically colorectal normal tissues，hyperplastic polyps，adenomas and adenocarcinomas were collected and retrospectively analyzed to establish diagnostic standards with MPM. Results：Through the investigation，we found the colorectal normal，hyperplastic polyps，adenoma and adenocarcinoma were different in the crypt opening，glandular structure，epithelial cells and collagen fibers. Besides，the cellular areas by quantitive analysis were 32.14 ± 1.96 μm2、32.69 ± 2.34 μm2、55.40 ± 4.63 μm2、71.83 ± 5.30 μm2、85.90 ± 11.50 μm2，respectively. P values were both of statistically significance. Conclusion：The real-time multiphoton microscopic imaging can be used to classify and grade the colorectal diseases safely and effectively with high resolution. It is potential to become the new generation of optical technique via integrating with the endoscopes in the future.