In this paper, to improve the control and optimization of the bio-fermentation process, rapid and non-destructive detection of glycerol and butanol was studied. To this end, 80 samples of glycerol and butanol were scanned and analyzed by means of near-infrared spectroscopy. Furthermore, in order to improve the predictive ability of the model, five quantitative calibration methods, namely, the partial least squares method, interval partial least squares method, forward interval partial least squares method, backward interval partial least squares method, and minimum forward interval method were used to establish the near-infrared detection model for glycerol and butanol content. The models were then analyzed and compared. The results showed that the model established by means of the backward interval partial least squares method was effective, and the correlation coefficients of the single component solution of glycerol and butanol were 0.99932 (glycerol) and 0.98843 (butanol), respectively. To measure the content of glycerol and butanol in the glycerol fermentation broth, a monitoring platform for glycerol and butanol concentrations was established. The correlation coefficients were found to be 0.99074 (glycerol) and 0.99261 (butanol), respectively. The results show that the NIR rapid detection model demonstrates excellent performance with regards to accuracy and rapidity and provides a new detection method for the rapid detection of glycerol and butanol in the fermentation industry.