Abstract: This study aims to compare various serum biochemical indices and the composition of serum metabolites in buffaloes（Bubalus bubalis） during lactation and dry periods. Additionally, it seeks to investigate the correlation between serum biochemical indices and metabolites to enhance our understanding of the regulatory mechanisms governing nutritional metabolism in buffaloes. The study selected nine healthy buffaloes in the lactation period（the 100th to 200th day of lactation） and nine in the dry period（the 30th to 60th day after stopping breastfeeding）. Two serum samples were collected from each buffalo. One of the collected serum samples from each buffalo was analyzed using a biochemical analyzer to quantify 20 indices. The other sample was subjected to ultra-high performance li-quid chromatography-tandem mass spectrometry（UPLC-MS/MS） to identify and quantify endogenous metabolites in the buffalo serum. Among the biochemical index detection results, the glutamic-oxaloacetic transaminase/glutamic-pyruvic transaminase（P＜0.05） and triglycerides（P＜0.01） in the lactation period were significantly lower than those in the dry period, indicating that the utilization efficiency of esters in buffalo during lactation was higher. Cholesterol（P＜0.01）, low-density lipoprotein（P＜0.01）, high-density lipoprotein（P＜0.001）, urea nitrogen（P＜0.01）, glucose（P＜0.01）, and immunoglobulin A（P＜0.000 1） were significantly higher in the lactation period than those in the dry period, indicating that the substance metabolism of buffalo during lactation was higher. The metabolomic analysis of the serum revealed that a total of 183 metabolites exhibited significant differences between the two periods under study（P ＜ 0.05, VIP ＞ 1）. These metabolites were enriched in 8 significantly different metabolic pathways, including D-glutamine and D-glutamate metabolism, arginine and proline metabolism, tryptophan metabolism, porphyrin and chlorophyll metabolism, glycine, serine and threonine metabolism, histidine metabolism, biosynthesis of unsaturated fatty acids and nitrogen metabolism（P ＜ 0.05）. These results show that during the lactation period, the nutrients absorbed by buffalo are mainly used to participate in the synthesis of various amino acids and the synthesis of fatty acids.