The objective of this study was to investigate the impact of partially substituting whole corn silage (CS) with sweet sorghum silage (SS) on serum metabolites in dairy cows employing by Liquid chromatography-mass spectrometry (LC-MS). Thirty mid-lactation Holstein dairy cows (112 ± 24.04 days in milk) were divided into the control group (CON) received 40% CS and 0% SS, Group1 received 30% CS and 10% SS, while Group2 received 20% CS and 20% SS, respectively. Serum samples were collected on day 45 of the formal experiment and subjected to LC-MS analysis to detect metabolites. The results revealed that 29 different metabolites were obtained in both CON and Group1; however, 22 metabolites in Group1 showed up-regulation compared to those in CON including N-Methylhydantoin, Betaine, Isoleucine, etc. The levels of 7 metabolites, including L-Kynurenine, Nandrolone, and Testosterone propionate, were found to be down-regulated. Sixteen metabolites were detected in the serum of Group2 and CON dairy cows. Compared with CON, Group2 exhibited up-regulation of 13 metabolites such as Benzaldehyde, Linoleic acid, Phe-Glu. Conversely, three metabolites including Triglycerides (TG), Uric acid and 5-Hydroxyindole-3-acetic acid showed down-regulation. A total of 20 differential metabolites were identified in Group1 and Group2. Among these, 14 metabolites such as 2-Ethylacrylic acid, Succinic acid, and Glutamic acid were up-regulated while 6 metabolites like Pyridoxine, Phosphocholine, and Quinidine showed down-regulation. These differentially expressed metabolites are primarily associated with various pathways including Aminoacyl-tRNA, Arginine, Phenylalanine and tryptophan biosynthesis, Citrate cycle etc. In conclusion, substituting up to 50% of CS with SS had no negative effects on serum metabolites, indicating that SS can partially replace CS in the diets of high-producing lactating dairy cows without adding extra grain, when diets are fed for a short time under our feeding experimental conditions.