The autofluorescence spectroscopy of biological tissues is a powerful tool for non-invasive detection of tissue pathologies and evaluation of any biochemical and morphological changes arising during the lesions’ growth. To obtain a full picture of the whole set of endogenous fluorophores appearing in the gastrointestinal (GI) tumors investigated, the technique of excitation-emission matrix (EEM) development was applied in a broad spectral region, covering the ultraviolet and visible spectral ranges. We could thus address a set of diagnostically-important chromophores and their alterations during tumor development, namely, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavins, porphyrins, while hemoglobin’s absorption influence on the spectra obtained could be evaluated as well. Comparisons are presented between EEM data of normal mucosae, benign polyps and malignant carcinoma, and the origins are determined of the fluorescence signals forming these matrices.