Background Unreasonable use of antibiotics in animals is a major concern and will remain so, thus affecting people's health. However, the application of plant extracts can better solve this problem. Objectives The purpose of this study was to study the effect of Moringa leaf flavonoids on the production performance, immunity, and rumen fermentation of dairy cows. Methods Nine Holstein multiparous cows (average weight: 550 kg; days of lactation: 150 ± 6 days) were used in the experiment, using a 3 × 3 Latin square design. Cows were divided into three groups, each of which was supplemented with 0, 50, or 100 mg/body weight (BW) Moringa oleifera leaf flavonoids. Each experimental period consisted of three periods of 21 days, and the prefeeding period lasted 15 days. Results Our results indicated that supplementation with Moringa leaf flavonoids increased the protein content and decreased the number of somatic cells in milk; had little effect on the biochemical indices of blood, the rumen fermentation, and serum biochemical indicators; and improved the activity of antioxidant enzymes, the antioxidant capacity, and immunity. Conclusions Addition of 50 mg/BW Moringa leaf flavonoids to cow enhanced the antioxidant and immunity capacity in dairy cows but did not affect physiological levels of common biochemical parameters in blood or fermentation parameters in rumen.