The influences of ultrasound‐assisted, pharmacopeia, and supercritical fluid extraction methods on bioactive compounds and biological activities of propolis were evaluated. Results showed that propolis extracted by ultrasound‐assisted method contained more phenolic compounds, and showed the highest total phenolic content (245.84 ± 6.41 mg GAE/g DW), total flavonoids content (198.82 ± 5.74 mg RE/g DW), and stronger in vitro antioxidant activity (DPPH·: 1.03 ± 0.04 mmol Trolox/g DW, ABTS+·: 2.19 ± 0.05 mmol Trolox/g DW, and FRAP: 1.48 ± 0.12 mmol FeSO4/g DW) than those of pharmacopoeia and supercritical fluid methods. A total of 36 phenolic compounds were identified in propolis. Among them, quercetin, quercetin‐3‐methyl‐ether, kaempferol, isorhamnetin, luteolin‐methyl‐ether, and quercetin‐7‐methyl‐ether could only be found in ultrasound‐assisted and pharmacopoeia methods. Moreover, the phenolic compounds had the similar metabolic pathways in rats and were mainly metabolized by sulfation and glucuronidation pathways. Additionally, ultrasonic‐treated propolis have good in vivo antioxidant activity and could repair D‐galactose‐induced oxidative damage in rats. Therefore, ultrasound‐assisted method could replace pharmacopeia method to be considered as bioactive compounds extraction from propolis, taking into consideration of yield, short extraction time, and high antioxidant activity.