Abstract: Hydrolysates derived from processing by-products of silver carp (Hypophthalmichthys molitrix), such as fish heads and fish bones, have been showed to promote probiotic growth, but current research on universal culture media for multiple strains remains insufficient. To develop a universal medium for multiple bacterial strains, enhance the high-value utilization of silver carp by-products, and promote the green development of the probiotic industry, we took silver carp processing by-products as raw materials, prepared protein hydrolysates via enzymatic hydrolysis, optimized the enzymatic hydrolysis conditions and nitrogen source substitution ratio, as well as explored the effects of the hydrolysates on the growth and metabolism of six probiotic strains (Lactobacillus acidophilus NCFM, Bifidobacterium animalis subsp. lactis BB-12, B. animalis subsp. lactis Bi07, L. acidophilus L01, Lacticaseibacill casei L18, L. rhamnosus LGG). The results show that the best effect was achieved when the hydrolysate obtained by papain hydrolysis for 1 h replaced 55% of the nitrogen source in MRS (deMan, Rogosa, Sharpe) medium (55%-N group); except for LGG, the absorbance at 600 nm (OD₆₀₀) of the other strains increased significantly (p<0.05), with no late-stage inhibition. Untargeted metabolomic analysis indicates that the 55%-N group maintained metabolic balance of different bacterial strains at different growth stages through the linkage of multiple pathways to enhance bacterial activity, among which taurine metabolism could improve antioxidant capacity, drive the conversion of pyruvate to lactic acid, and synergize with other pathways to reduce environmental pH value for promoting growth. In summary, the study confirms that silver carp by-product hydrolysate serves as an effective alternative to traditional nitrogen sources, which provides a new strategy for developing low-cost and high-efficiency multi-strain fermentation media.