Comparative optical study of biofluids (serum, urine, hemodialysate) by concentration change of endogenous visible fluorescence substance (VFS) has been carried out. Biofluids were collected from chronic kidney diseases (CKD) patients (Pts) as well as from healthy controls (HCs). Excitation/emission spectra are similar for all samples of biofluids differing only in intensity, which is higher for CKD Pts. Strong similarity enables the study of given biofluids from a united physical platform, proposed earlier, i.e., as nanoparticles approach. Specific spectral redistribution of visible fluorescence (VF) intensity as a result of dilution is revealed. The concentration change of VFS by dilution of samples manifests in nonlinear dependences in the scales “VF intensity–concentration" for serum and urine and in perfect linearity for hemodialysate (HD). The latter fact can be used in monitoring of hemodialysis procedure.