Agarwood essential oil is prized for its elegant aroma and pharmacological properties
however, the traditional hydrodistillation method suffers from inefficiencies, constraining the industrial potential of agarwood. We proposed an ultrasonic-assisted laccase synergistic pretreatment technique that enhanced extraction throughput by 70.90 % compared to the traditional method by facilitating pore formation in agarwood and expediting the release of essential oil. The essential oil extracted using this method retained a similar aromatic profile to the traditional method but achieved a higher sesquiterpene oxide concentration (95.3 % vs. 89.6 %). Additionally, it exhibited notable improvements in antioxidant activity, acetylcholinesterase inhibition, and α-glucosidase inhibition, attributed to the increased affinity of sesquiterpene oxides for enzymes through hydrogen bonding, covalent interactions, and hydrophobic effects with amino acid residues. This approach not only maximises the efficiency of agarwood essential oil extraction and amplifies its bioactive properties, but also offers a sustainable and eco-friendly alternative, promoting the long-term vitality of the agarwood industry.