Autofluorescence (AF) spectra of colonic normal and adenocarcinoma tissues are measured under excitation of 337 nm and analyzed by multivariate curve resolution alternating least squares (MCR-ALS) method using non-negativity constraint. Collagen, nicotinamide adenine dinucleotide hydrate (NADH) and elastin are identified as the main contributing biomedical components. Fisher’s discrminant anlysis (FDA) on the concentration profiles of the principle components (PCs) shows acceptable sensitivity, specificty and accuracy for discrminanting the adenocarcinoma tissues from the normal tissues. MCR-ALS is a powerful tool for characterzing the spectra profiles of the main biochemical components in neoplasm transformation.