Abuse of antibiotics has led to excessive amounts of antibiotic residues in food and environment, thus enhancing pathogenic bacterium resistance and threatening human health. Therefore, searching and developing safe and green antibiotic alternatives are necessary. In this study, an Artemisia argyi leaf polysaccharide (AALP) fraction was extracted and analyzed. Chemical composition analysis showed that the carbohydrate, uronic acid, protein, and polyphenol content in AALP were 68.3 % ± 4.13 %, 9.4 % ± 0.86 %, 1.79 % ± 0.27 %, and 0.16 % ± 0.035 %, respectively. Chromatographic results suggested that AALP contained rhamnose, arabinose, glucosamine, galactose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid in a molar ratio of 9.26, 1.35, 1.18, 3.04, 48.51, 2.33, 31.26, 3.93, and 9.08; the weight average molecular weight, number average molecular weight, and polydispersity of AALP were 5.41 kDa, 4.63 kDa, and 1.168, respectively. Fourier transform infrared spectroscopy indicated that AALP constituted the polysaccharide-specific groups of CH, CO, and OH. Meanwhile, AALP showed a dose-dependent inhibitory effect on Staphylococcus aureus in the inhibition zone assay, and the minimal inhibitory concentration was 1.25 mg/mL. Furthermore, AALP disrupted the cell wall, depolarized the inner membrane potential, and inhibited the activities of succinate dehydrogenase and malate dehydrogenase in S. aureus.