The glucoamylase fermentation was investigated with Fourier transform infrared spectroscopy. During the cultivation, the variation of carbon and nitrogen source can be reflected from the infrared spectra. In most cases, the complex media are used to cultivate cells or bacteria. It is difficult to construct calibration samples with synthetic mixtures which can represent the composition of real broth. The infrared spectral data of the real broth and off-line chemical analysis results were used to establish the calibration model. The model is successfully used to predict the concentrations of total sugar, reduced sugar, and nitrogen in real broth. The correlation coefficients between the infrared analysis results and chemical analysis are 0.999, 0.990 and 0.974 for total sugar, reduced sugar and nitrogen, respectively. The bacteria, culture and operating condition are relatively stable during the practical productive processes. The method can be used to monitor the industrial processes.