Objective To comparatively evaluate the effects of three extraction methods, i.e., steam distillation (SD), enzyme-assisted steam distillation (EHSD), and ultrasound-assisted steam distillation (UASD), on the extraction efficiency, chemical composition, and bioactivity of Citrus medica L. var. sarcodactylis (Guangfoshou) essential oil.Methods Guangfoshou essential oil was extracted using SD, EHSD, and UASD. The chemical constituents of the essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS). Cluster heatmap analysis was employed to compare differences in essential oil composition obtained by the three extraction methods. Antioxidant activity was assessed based on total antioxidant capacity, DPPH radical scavenging activity, and hydroxyl radical scavenging activity.Results The yields of Guangfoshou essential oil obtained by the three methods, in descending order, were UASD (1.750%)>EHSD (1.252%)>SD (1.250%). A total of 38 components were identified by GC-MS, mainly olefinic compounds, among which 18 were common to all three methods. Among the common components, D-limonene (45%~47%) and γ-terpinene (28%~30%) exhibited the highest relative contents. The differential components included six compounds: β-pinene, α-pinene, D-limonene, tert-butyl peroxycyclopentanecarboxylate, geranylacetone, and (Z)-citral. KEGG pathway enrichment analysis of these differential components showed that the top 20 pathways ranked by P-value were mostly closely related to oxidative stress. Antioxidant results indicated that, within the tested concentration range, the total antioxidant capacity of essential oils obtained by the three methods decreased in the order UASD>SD>EHSD. The DPPH radical scavenging activity followed the order UASD (IC₅₀=31.15 mg/mL)>EHSD (IC₅₀=32.82 mg/mL)>SD (IC₅₀=34.74 mg/mL), while hydroxyl radical scavenging activity followed the order UASD (IC₅₀=3.82 mg/mL)>SD (IC₅₀=9.14 mg/mL)>EHSD (IC₅₀=9.43 mg/mL).Conclusion UASD can be considered the optimal method for the extraction of Guangfoshou essential oil.