The fluorescence signal increase in deuterated water - D2O is relevant to many methods used in chemical and biological research. We performed fluorescence spectroscopy measurements combined with evaluations of fluorescence-based methods to describe and quantify the practical impacts of D2O on chemical and biological samples. Several novel features are observed in fluorescence spectra and life-time decay curves of molecules including fluorescence probes and labels that are used frequently in biomolecular labelling and bioimaging. The observed characteristics suggest that D2O enhances the fluorescence signal due to the exchange of labile hydrogen on a fluorophore for heavier deuteron and the reduction of energy transfer from excited fluorophores to H2O molecules. Furthermore, performed quantifications approve that D2O increases significantly the detection signal of all examined fluorescence-based approaches where the brightness of fluorophores and sample viability are crucial.