To explore the influence of milk powder storage by-products on the probiotic activity, Lacticaseibacillus paracasei JY025 fortified milk powder was used to evaluate the change of bacterial activity and the accumulation of Maillard and lipid oxidation by-products during long-term storage. Based on the above results, this study further clarifies the effect mechanism of milk powder by-products on the metabolism and transcriptional response of Lacticaseibacillus paracasei JY025. The results of the strain JY025 growth ability and its metabolic ability of various carbon sources under different by-product interventions indicated that the Maillard by-products (higher concentrations) and the lipid oxidation by-products inhibit the growth metabolic of Lactobacillus. The transcriptomic analysis of strains under different interventions further suggested the intervention of Maillard by-products with a specific concentration improve the ability of Lactobacillus to utilize carbohydrates, which helps to provide additional energy and improve the tolerance of Lactobacillus to lipid oxidation by-product stress.