This study was conducted to explore the effects of organic trace elements on milk composition, serum trace element concentrations, antioxidant and immune function, and metabolites in dairy cows. Thirty Holstein cows with similar body condition scores (3.5 ± 0.25 points) and parity were randomly divided into a control group (C), a 50% organic group (O_50), and a 100% organic group (O_100), with 10 cows in each group. The C group was fed a basal diet supplemented with 3% inorganic trace element premix, the O_50 group received 1.5% inorganic and 1.5% organic trace element premix, and the O_100 group received 3% organic trace element premix. Organic trace elements were supplemented from 21 days postpartum for 90 days. The results showed that milk protein concentration showed a linear response to organic trace element supplementation, with the highest value in the O_50 group (P < 0.05). Lactose concentration showed an increased quadratic effect (P < 0.01), whereas total solids (TS) and solid-not-fat (SNF) concentrations decreased linearly and quadratically. Serum selenium (Se) concentration increased linearly with increasing organic trace element supplementation (P < 0.05). Malondialdehyde (MDA) concentration decreased linearly (P < 0.05), whereas serum immunoglobulin A (IgA), superoxide dismutase activity, and immunoglobulin G (IgG) concentration increased significantly (P < 0.05). Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis identified 70 and 76 differential metabolites in the O_50 and O_100 groups, respectively, compared with the C group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that these metabolites were mainly involved in inflammatory mediator regulation, lysine degradation, sphingolipid metabolism, cytochrome P450-mediated drug metabolism, and ABC transporters. In conclusion, dietary supplementation with organic trace elements improved milk composition, antioxidant capacity, immune function, and serum metabolic profiles in dairy cows.