In this work, the use of excitation-emission fluorescence matrices (EEMs) has been addressed to differentiate two kinds of cheeses with protected designations of origin (PDO), “Torta del Casar” and “Queso de la Serena”. The fluorescence signals were recorded directly on cheese samples during the ripening process, by using a fiber optic assembled to the fluorescence spectrometer, and the data were subjected to analysis by the multivariate algorithm parallel factor analysis (PARAFAC). PARAFAC analysis identified the presence of three fluorophores in both cheeses. The scores of the two first components, attributed to the presence of tryptophane, decreased as the ripening time increased. However, the third component, related to retinol (vitamin A), remains almost constant along the ripening period. EEMs of the samples of the two types of cheese were similar in the initial days of the maturation periods. However, when only the samples from day 36 onwards were considered, it was possible to differentiate them with high precision by PARAFAC in combination with linear discriminant analysis (LDA). In addition, a model was built for the quantification of vitamin A and α-tocopherol (vitamin E), by using the EEMs combined with the multivariate unfolded partial least squares (UPLS) algorithm. •Excitation-emission matrices were used to monitor the ripening process in cheeses.•The cheese fluorescence intensity decreased as the maturation time progressed.•Three fluorophores were identified by using parallel factor analysis algorithm.•Linear discriminant analysis allowed to discriminate between both types of cheeses.•U-PLS algorithm was apply in order to predict the level of vitamin A and E.