The monitoring and evaluation of colorectal adenoma–carcinoma sequence during endoscopy are important for endoscopic resection of precursor lesions to disrupt the adenoma–carcinoma sequence and halt progression to invasive neoplastic disease. In this study, multiphoton microscopy (MPM) was used to identify different stages during the development of colorectal adenocarcinoma including adenoma with low-grade and high-grade dysplasia, and adenocarcinoma invading the submucosa. It was found that by combining two-photon excited fluorescence (TPEF) imaging and second harmonic generation (SHG) imaging, MPM can reveal the morphological changes of the epithelial cells and glands, identify the invasive position and depth of atypical glands and quantitatively describe the change of the cellular nucleus and the nuclear-to-cytoplasmic ratio during the stepwise progression of colorectal adenocarcinoma. These are important pathological findings for pathologists when diagnosing colorectal lesions. With the advancement of a compact and flexible multiphoton endoscope for in vivo imaging and clinical applications, MPM has the potential to provide immediate histological diagnosis for the monitoring and evaluation of the colorectal adenoma–carcinoma sequence during endoscopy.