Abstract-Rice wine, the traditional Japanese alcohol, contains ethanol and glucose, whose concentrations are evaluated to manage their quality. Recently, infrared techniques have been introduced to rice wine measurement, and absorption spectra are analyzed to monitor rice wine components during fermentation. However, it is difficult to precisely evaluate ethanol and glucose concentrations because of an overlapping of their absorption peaks. This paper proposes a spectral separation method using a single absorption band for evaluating the ethanol and glucose concentrations in rice wine. We evaluate their concentrations by separating a mixed spectrum into ethanol and glucose spectra. The simulation results showed a decrease in the estimation errors of ethanol and glucose concentrations from 5.40±5.41 %(v/v) and 0.73±0.73 g/dl, respectively, using regression analysis (RA) to 0.46±0.65 %(v/v) and 0.40±0.57 g/dl, respectively, using the proposed method.