The consumption of herbal teas has become popular in recent years due to their attractive flavors and outstanding antioxidant properties. The Five-Golden-Flowers tea is a herbal tea consisting of five famous edible flowers. The effects of microwave-assisted extraction parameters on the antioxidant activity of Five-Golden-Flowers tea were studied by single-factor experiments, and further investigated using response surface methodology. Under the optimal parameters (53.04 mL/g of solvent/material ratio, 65.52 °C, 30.89 min, and 500 W), the ferric-reducing antioxidant power, Trolox equivalent antioxidant capacity, and total phenolic content of the herbal tea were 862.90 ± 2.44 µmol Fe2+/g dry weight (DW), 474.37 ± 1.92 µmol Trolox/g DW, and 65.50 ± 1.26 mg gallic acid equivalent (GAE)/g DW, respectively. The in vivo antioxidant activity of the herbal tea was evaluated on alcohol-induced acute liver injury in mice. The herbal tea significantly decreased the levels of aspartate aminotransferase, total bilirubin, and malonaldehyde at different doses (200, 400, and 800 mg/kg); improved the levels of liver index, serum triacylglycerol, and catalase at dose of 800 mg/kg. These results indicated its role in alleviating hepatic oxidative injury. Besides, rutin, chlorogenic acid, epicatechin, gallic acid, and p-coumaric acid were identified and quantified by high performance liquid chromatography (HPLC), which could contribute to the antioxidant activity of the herbal tea.