In the present study, the effect of unilateral (ULC) and bilateral castration (BLC) on metabolomics in yellow cattle was conducted. The current trial was comprised of 120 days of feeding following castration, after completion of the trial the blood samples were collected from all groups for metabolomics analysis. The results displayed 46 important metabolites were found when BLC compared to the CON group (VIP >1P < 0.05), such as GPCho (22:6/18:2), 7,8-Dihydrobiopterin, phosphocholine, phosphatidylcholine (PC), Butyryl-L-carnitine and 5-Hydroxyindoleacetic acid etc. The KEGG pathway analysis showed that BLC affects the 9 metabolic pathways in the yellow cattle serum when compared with the CON group. Biomarker analysis showed that the metabolites PC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)/18:2(9Z,12Z)),PC(20:4(8Z,11Z,14Z,17Z)/P-18:0),PC(18:0/22:5(4E,7E,10E,13E,16E))[U],PC(17:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))and butyryl-L-carnitine etc. were the serum biomarkers between BLC and CON groups. In addition, when we compared the differential metabolites between ULC and BLC groups, 58 differential metabolites were found. KEGG pathway analysis showed that the metabolome pathway between ULC and BLC cattle affects 12 pathways in the serum of the yellow cattle. The BLC group cattle had a higher serum concentration of phosphatidylcholine and LysoPC enriched in the Glycerophospholipid metabolism pathway and a higher serum concentration of PC(16:0/20:4(5Z,8Z,11Z,14Z)) enriched in the Arachidonic acid and Linoleic acid metabolisms pathway, respectively. The Phospholipid Metabolites were the biomarkers in the serum of BLC cattle. Therefore, Castration enhanced the synthesis metabolism of unsaturated fatty acids and increased concentrations of unsaturated fatty acids, including linoleic acid (C18:2n6c), linolenic acid (C18:3n3), arachidonic acid (C20:2), docosahexaenoic acid (C22:6n3), in the fat tissue of cattle.