Fluorescence phenomena in fungi have been investigated only sparingly, despite long-standing evidence that many fungal metabolites exhibit autofluorescence. This study presents a standardized fluorescence spectroscopy method for analyzing extracts from four cultivated mushrooms (Pleurotus eryngii, Hypsizygus tessulatus, Hericium coralloides, and Lentinula edodes), with the aim of identifying interspecific differences and developmental stage-related variations. Sporophore samples were extracted with ethanol and n-hexane and analysed by fluorescence emission spectroscopy. Significant differences in fluorescence intensity and spectral distribution among the different species have been observed. H. coralloides and P. eryngii displayed the strongest fluorescence, in spines and gills, respectively. Comparison of different growth stages of P. eryngii indicated that fluorescence intensity increased significantly as the mushroom reached its fully mature sporulating stage. This increase might be correlated with the ability of spores to emit fluorescence. In all experiments, n-hexane was less efficient than ethanol in extracting fluorophores (fluorescent pigments). A notable exception concerned the gills of P. eryngii for which the emission band was observed at lower wavelengths (420 nm). In contrast, ethanol extracts showed stronger fluorescence with emission peaks generally situated in the blue range at around 450 nm. These findings demonstrate that fluorescence spectroscopy provides a rapid and reproducible analytical method for characterizing fungal tissues with potential applications in fungal taxonomy, metabolite screening, and quality control in mushroom cultivation.