Plant essential oil (EO) is an important natural active product, and the development of efficient and green EO separation technique has been one of the research hotspots. In this work, surfactant-enzyme pretreatment in high-solid system followed by microwave-assisted hydrodistillation (SEHS-MHD) was developed for the isolation of EO from Mosla chinensis Maxim. Through kinetic curve fitting, pretreatment of raw materials with combined enzyme (cellulase and hemicellulase of 1:1 in activity value) and surfactant (tween-20) in high-solid system (20% solid content) could provide a higher yield of EO. Compared with other reference methods, the yield of EO obtained by SEHS-MHD was significantly increased, which was mainly due to the synergistic interaction of enzyme and surfactant in the high-solid system. The results of gas chromatography-mass spectrometer analysis showed that the main components of M. chinensis EO were thymol and carvacrol, the contents of which were 75.38% and 17.20%, respectively. Additionally, the inhibitory enzyme activity and antifungal activity of M. chinensis EO were evaluated, the results of which indicated that EO had no obvious inhibitory activity on alpha-amylase and acetylcholinesterase, but had spectral antifungal activity. In general, the SEHS-MHD developed in this work is a potential and powerful alternative to traditional techniques for the sufficient isolation of EO from natural plant and crop materials.