A simple, rapid and accurate mid-infrared (MIR) spectroscopic method for simultaneously determining the product (ethanol) content and the nutrient (sugar) content in plant-cell culture media was developed using a Fourier transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) accessory. We assessed the potential of this method by comparing it to a high-performance liquid chromatography (HPLC) method, and using the developed method to measure the ethanol and sugar contents simultaneously in liquid culture media with rice and tabacum cell suspensions, respectively. The experimental results suggest that the sugar consumption and ethanol production behaviors of the plant cell suspensions can be non-destructively and simultaneously monitored using the developed method. Furthermore, the spectroscopic method provided in this study could be developed into a technique that could be used to analyze the overall kinetics of the metabolism of the plant cell suspensions.