Acute ischemic stroke (AIS) is characterized by a sudden blockage of one of the main arteries supplying blood to the brain, leading to insufficient oxygen and nutrients for brain cells to function properly. Unfortunately, metabolic alterations in the biofluids with AIS are still not well understood. In this study, we performed high-throughput target metabolic analysis on 44 serum samples, including 22 from AIS patients and 22 from healthy controls. Multiple reaction monitoring analysis of 180 common metabolites revealed a total of 29 metabolites changed significantly (VIP>1, P <0.05). Multivariate statistical analysis unraveled a strikingly separation between AIS patients and healthy controls. Comparing AIS with Control group, the contents of argininosuccinic acid, beta-D-glucosamine, glycerophosphocholine, L-abrine, and L-pipecolic acid were down-regulated in AIS patients. 29 out of 112 detected metabolites, enriched in aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, lysine degradation, phenylalanine, tyrosine and tryptophan biosynthesis metabolic pathways. Collectively, these results will provide a sensitive, feasible diagnostic prospect for AIS patients.