This study utilized transient autofluorescence spectroscopy to measure the autofluorescence lifetimes of normal and cancerous colorectal tissues at five optimal excitation-emission wavelengths. The performance of these lifetimes for classifying normal and cancerous tissues was evaluated. The results indicate significant differences in autofluorescence lifetimes between normal and cancerous colorectal tissues at wavelengths of 340~380 nm, 340~470 nm, 405~515 nm, and 405~635 nm. Jouden index analysis determined classification thresholds of 6.153 ns, 5.847 ns, 5.130 ns, and 6.414 ns, respectively. The receiver operating characteristic (ROC) curve analysis indicated that the peak diagnostic performance was achieved at 405~635 nm, with sensitivity, specificity, and area under the curve (AUC) values of 85.7%, 100%, and 0.908 (P<0.001, 95% CI: 0.78~1.00), respectively. The findings suggest that autofluorescence lifetime measurements serve as a promising diagnostic biomarker for colorectal cancer, potentially aiding in its early detection.