By using four labels of the 3-hydroxyflavone family displaying selective sensitivity to hydrogen bond (HB) donors and poor response to other polar molecules, we developed an approach for measuring local water concentration [H(2)O](L) (or partial volume of water: W(A) = [H(2)O](L)/55.6) in the label surrounding both in solvent mixtures and in biomolecules by the intensity ratio of two emissive forms of the label, N*/T*. Using a series of binary water/solvent mixtures with limited preferential solvation effects, a linear dependence of log(N*/T*) on the local concentration of HB donor was obtained and then used as a calibration curve for estimating the W(A) values in the surroundings of the probes conjugated to biomolecules. By this approach, we estimated the hydration of the labels in different peptides and their complexes with DNAs. We found that W(A) values for the label at the peptide N-terminus are lower (0.63-0.91) than for free labels and depend strongly on the nature of the N-terminal amino acid. When complexed with different DNAs, the estimated hydration of the labels conjugated to the labeled peptides was much lower (W(A) = 0-0.47) and depended on the DNA nature and linker-label structure. Thus, the elaborated method allows a site-specific evaluation of hydration at the surface of a biomolecule through the determination of the partial volume of water. We believe the developed procedure can be successfully applied for monitoring hydration at the surface of any biomolecule or nanostructure.