Flavonoids extracted from Pandanus amaryllifolius Roxb. are recognized for their superior bioactivity and potential applications in functional foods and nutraceuticals. The extraction process, composition, and antioxidant activity of flavonoids from P. amaryllifolius Roxb. leaves were optimized, characterized, and evaluated, respectively, by employing ultrasound-microwave-assisted extraction; optimal conditions were determined using single-factor experiments combined with response surface methodology. Flavonoid composition was analyzed by high-performance liquid chromatography-tandem mass spectrometry; antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS+) free radical scavenging, reactive oxygen species (ROS) scavenging, and intracellular antioxidant assays. The results indicated that 74% (V/V) ethanol concentration, a liquid-to-solid ratio of 10.5:1 mL•g-1, an extraction temperature of 61 ℃, a microwave power of 530 W, an ultrasonic power of 350 W, an ultrasound-microwave treatment time of 20 min, and a total extraction time of 25 min were optimal extraction conditions. Under these conditions, the extraction yield reached 12.14 mg•g-1. In total, 22 flavonoids were identified in the extract, including quercetin, rutin, and nobiletin. The flavonoids extracted from P. amaryllifolius Roxb. leaves exhibited potent antioxidant capacity, effectively scavenging DPPH and ABTS+ free radicals, with IC50 values of 54.4 and 141.3 μg•mL-1, respectively. Additionally, they exhibited significant potential in mitigating radical-induced quenching and reducing intracellular ROS generation. This experiment provides technical support for the extraction and application of flavonoids from P. amaryllifolius Roxb. leaves.