Response surface methodology was used to optimize the ultrasonic-assisted extraction (UAE) of total flavonoids (TF) and total phenols (TP) from Berberis kongboensis fruit. Single-factor experiments were conducted with the ultrasonication power, liquid–solid ratio, ethanol concentration, extraction temperature, and extraction time. A Plackett–Burman design was used to identify the main factors affecting the extraction rates of TF and TP. A Box–Behnken design was used to further optimize the ethanol concentration, solvent/sample ratio, and extraction temperature. Under the optimum conditions, the ethanol concentration of 34.67%, solvent/sample ratio of 38.44 mL/g, and extraction temperature of 46.53 °C, the yields of TF and TP were 107.92 mg rutin equivalents/g and 4.86 mg gallic acid equivalents/g, respectively. The extract from UAE had a better yield and higher antioxidant activities in vitro than that obtained by conventional solvent extraction. The B. kongboensis fruit is rich in flavonoids and phenolics, and it has potential application value in the food industry.