A simple, rapid and accurate evaluation of the sugar uptake rate of suspended plant cells from culture media was developed with the predicted sugar contents measured by mid-infrared spectroscopy using a Fourier transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) accessory. We performed plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) in culture media, which had various combinations of glucose, fructose and sucrose concentrations at the initial stage, and measured simultaneously each sugar content in the medium by the FT-IR/ATR method. By applying a logistic function to the predicted sugar contents and cell density in the medium during cultivation, the specific sugar uptake rates by the suspended TBY-2 cells were easily and continuously obtained. Thus the kinetic sugar uptake phenomena by the TBY-2 cells were well confirmed overall using the developed method. Additionally it was found that the fraction of sucrose of the initial total sugar content might kinetically affect the sugar uptake process and cell growth. Also, the relationship between the nondimensional cell density and sucrose content could be classified into three groups on the basis of the initial fraction of sucrose.