In this paper, method of monitoring bacillus subtilis fermentation process using attenuated total reflectance mid infrared (ATR-MIR) spectroscopy is explored. The bacillus subtilis fermentation produces poly-glutamic acid which is widely used. The routine measure of fermentation broth includes glucose, glutamic acid, poly-glutamic acid, and biomass. Traditional method of monitoring fermentation process is based on biosensor or spectrophotometer, which is laborious and time consuming. ATR-MIR spectroscopy is used in the fermentation process, which is efficient and multivariate can be obtained simultaneously in one measurement. Calibration method is addressed take into account the peculiarity of fermentation liquid, such as solvent concentration change correlation in fermentation batch. Using PLS several concentrations of solvents are get and the fermentation process can be monitored timely. The experiment results show that spectroscopy is a feasibility method in the poly-glutamic acid producing bacillus subtilis fermentation process.