The aim of this study was to investigate the antioxidant effect of alpha-lipoic acid (α-LA) in dairy cows and its metabolic mechanism. Thirty Holstein cows weighing 550 ± 25 kg, 200 ± 15 days of lactation and calving 2–3 times were randomly divided into three groups, ten cows in each group. Different doses of α-LA were added based on body weight: 0 (CTL), 30 (LA-L) and 60 (LA-H) mg/kg per head per day; 7 days adaptation period, 30 days formal period. Milk production was recorded daily during the test period. Milk and blood samples were collected on the last day. ELISA kits and automatic biochemical analyser were used to detect the indicators in blood; serum metabolites were detected and analysed by non-target metabolomics. The results of the study showed that the addition of α-LA significantly increased milk yield; blood concentrations for HDL, triglyceride, cortisol and triiodothyronine were significantly elevated; and levels of glutathione reductase and nitric oxide synthase were significantly reduced in LA-L group as compared to CTL group. The concentrations of IL-1β, IL-2, TNF-α, IgG and IgA were significantly higher after supplementation with α-LA. Metabolomics analysis revealed 13 and 15 differential metabolites each in positive or negative modes. Methylmalonic acid levels were significantly higher following α-LA supplementation compared to CTL group, as were D-lactose, D-maltose and oleanolic acid levels in LA-L group. In summary, α-LA can enhance milk production, improve antioxidant capacity and immunity, and is more beneficial for animal production and economic benefits at 30 mg/kg.