Objective: To investigate the metabolic profiles of blood amino acids, carnitines, and succinylacetone in neonates, analyze their influencing factors, and identify differential metabolites and metabolic pathways between preterm and full-term infants, thereby providing a basis for clinical intervention in preterm infants. Methods: A total of 19 599 neonates delivered at medical and healthcare institutions in Huai' an City, Jiangsu Province from July 2023 to June 2024 were enrolled. According to gestational age, they were divided into two groups: the preterm group (28 weeks≤gestational age<37 weeks, n=1 107) and the full-term group (37 weeks ≤ gestational age < 42 weeks, n=18 492). Dried blood spot samples were collected within 7 days after birth. Tandem mass spectrometry was used to measure blood metabolic parameters. Spearman rank correlation analysis was applied to examine the correlations among sex, gestational age, birth weight, and metabolic parameters. The t test and partial least squares discriminant analysis (PLS-DA) were used to identify significantly different metabolites between the two groups, and the intersection of the differential metabolites obtained by the two methods was selected as potential biologically significant biomarkers. Pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify differential metabolic pathways. Results: Multiple blood amino acids and carnitine metabolic parameters were significantly correlated with neonatal sex, gestational age, and birth weight(P<0.05). A total of 18 significantly different metabolites were identified between preterm and full-term infants. Of them, 8 metabolites were upregulated in the preterm group, including free carnitine (C0), arginine (Arg), and phenylalanine (Phe); 10 metabolites were downregulated, including alanine (Ala), glycine (Gly), and decanoylcarnitine (C10). These differential metabolites were involved in 17 metabolic pathways, including arginine and proline metabolism; phenylalanine, tyrosine and tryptophan biosynthesis; phenylalanine metabolism; biosynthesis of unsaturated fatty acids; fatty acid degradation; and fatty acid biosynthesis. Conclusion: Blood amino acid and carnitine levels in neonates are significantly correlated with sex, gestational age, and birth weight. There are distinct metabolic differences between preterm and full-term infants, which may affect amino acid and fatty acid metabolic processes.