Abstract: Nitrile hydratase (NHase) is one of the key enzymes in nitrile catabolism, which can catalyze the conversion of aliphatic nitriles or aromatic nitriles to the corresponding amides. Currently, NHase is used in the industrial production of acrylamide (AM) and niacinamide through the hydration of acrylonitrile (AN) and 3-cyanopyridine, respectively. Rhodococcus has been widely used in the biotransformation of AN due to their excellent properties such as high NHase activity, heat resistance and good stability in organic solvents. In this study, the effects of different concentrations of glucose and cobalt chloride on the synthesis of nitrile hydratase (NHase) were investigated. The results showed that the glucose mass concentration of 20 g/L and the cobalt chloride concentration of 0.050 g/L could effectively enhance the synthesis of NHase. Subsequently, a process physiology metabolic parameter collection and analysis system based on fermentation process off-gas mass spectrometer were established. Through multi-parameter correlation analysis, pH was identified as a key factor influencing the expression of NHase in Rhodococcus rhodochrous J1, and thus a strategy of continuous adding sodium gluconate during the fermentation process to control the pH was proposed. Compared to the original batch fermentation, the optimized process exhibited a total enzyme activity of NHase increased by 93.74%, the specific enzyme activity increased by 97.35% and the sugar conversion rate increased by 71.36% in continuous batch fermentation under the same bacterial concentration. This paper provides theoretical guidance for enhancing acrylonitrile hydratase production in industrial settings.