Abstract: To enhance the deep-processing value of highland barley, this study took starch degradation rate as the key indicator and optimized the fermentation process of highland barley water extract by Bacillus subtilis KC-4 through single-factor experiments and response surface methodology. The metabolic mechanism was further investigated via dynamic monitoring of fermentation and untargeted metabolomics. The results showed that the optimal fermentation parameters were as follows: initial pH8.0, solid-to-liquid ratio 1:15, loading volume 35%, inoculation amount 6.3%, temperature 37 ℃, and time 48 h. Under these conditions, the starch degradation rate reached 88.13%. During fermentation, the activities of α-amylase and protease increased continuously, promoting the degradation of starch and protein, accompanied by the accumulation of reducing sugars, free amino acids, total polyphenols, and total flavonoids, along with a significant improvement in antioxidant capacity. Untargeted metabolomics analysis revealed that amino acids and their derivatives were the main differential metabolites (accounting for 45.07%), and pathways such as D-amino acid metabolism and amino acid biosynthesis were significantly enriched. Accordingly, a synergistic and mutually reinforcing metabolic network of “protein degradation→amino acid supply→amylase synthesis→starch degradation→energy replenishment” was proposed, clarifying the internal mechanism by which KC-4 efficiently drives starch degradation and enhances product activity through carbon-nitrogen metabolic coordination. This study provides a theoretical basis and technical support for the high-value utilization of highland barley, and offers a new perspective for understanding the regulatory mechanisms of microbial fermentation.