Abstract: In this study, an efficient microwave-assisted hydrodistillation method was developed to separate the essential oil from Psidium guajava leaves. The main influencing factors of the process were optimized using single factor method. Under the optimized conditions, the yield of essential oil was 31.76 ± 1.45 mL·kg^-1, and the dominant components of Psidium guajava leaves essential oil were caryophyllene, calamenene A, nerolidyl acetate and humulene epoxide. Compared with traditional hydrodistillation method, the microwave-assisted hydrodistillation had high yield, short time consumption, and low power consumption. Microwave-assisted hydrodistillation is considered as a potentially effective and sustainable alternative technique for the separation of essential oils from plant matrix.