Microwave-assisted extraction (MAE) of polyphenols from hawthorn (Crataegus pinnatifida Bge.) fruit was investigated, and the best combination of extraction parameters was obtained using response surface methodology (RSM). The optimised procedure was achieved by soaking the sample powder with 83% ethanol for 90 min at solid–liquid ratio of 1–20, and then microwave irradiation for 13 min at power of 440 W. Under this condition, an extraction yield of 23.5% was obtained and the percentage of phenolic compounds in the extract reached 41.2%. The total content of polyphenols in hawthorn fruit was 96.9 ± 4.3 mg gallic acid equivalents per gram of dry weight measured by Folin-Ciocalteu reagent method, and the main phenolic compounds were determined as procyanidin B2, epicatechin, chlorogenic acid, procyanidin C1 and rutin. Biological studies showed that the extract possessed a strong inhibitory effect against DPPH, hydroxyl radicals and lipid peroxidation, as well as strong reducing power.