A ¹H NMR-based metabolomics approach was used to explore the impact of dietary Zn on the serum metabolic profile of Megalobrama amblycephala. Fish were fed diets containing graded levels of Zn (7.4, 32.1, and 332.4 mg/kg diet; supplied as ZnSO₄·7H₂O) for 12 weeks. We identified 52 metabolites in serum including 23 amino acids or intermediary metabolites, 16 organic acids, 4 sugars, 4 nucleic acids, 3 vitamins and 2 others. Multivariate data analyses (PLS-DA and VIP) showed dietary Zn affected the metabolic profile impartially and these metabolites (such as proline, lactate, glucose, alanine, lysine, glutamine, creatine, trans-4-hydrox-L-proline, taurine, serine, τ-methylhistidine, arginine, valine, methionine and mannose) were identified as charactisteristic metabolites. These metabolites are mainly increased in the groups (7.4 and 332.4 mg/kg diet) compared with the group (32.1 mg/kg), suggesting that Zn deficiency and excess inhibited amino acid and carbohydrate metabolism. In conclusion, proline, lactate and glucose were found to have potentials for being new biomarkers for Zn nutritional status of M. amblycephala.