An online concentration monitoring method was established by using attenuated total reflection ultraviolet (ATR-UV) coupled with a suitable partial least squares regression for a multi-solute crystallization system, namely, disodium 5'-inosinate (IMP) and disodium 5'-guanylate (GMP) in aqueous ethanol. The results indicated that the established method accurately predicted the concentration of IMP and GMP in the solution. The solubility data was measured at various temperatures and ethanol/water mass ratios, and the experimental data was well correlated by applying the modified Apelblat equation and modified Jouyban-Acree model. The implementation of cooling crystallization demonstrates that the developed calibration model is suitable for online concentration monitoring with reasonable accuracy and precision.