Rosemary (Rosmarinus officinalis L.) is an important cash crop, and its essential oil (EO) has been widely used in food, chemical, pharmaceutical and other industries. The efficient and green separation of rosemary essential oil (RO) can provide technical support for its application and realize the high-value utilization of rosemary resources. In this work, RO was separated by a modified homogenate-ultrasonic pretreatment followed by microwave hydrodistillation (HUP-MHD) technique. The influencing parameters involved in the homogenate, ultrasonic pretreatment and microwave hydrodistillation processes were screened and optimized by single factor experiment and further response surface experiment (BBD), which derived the RO yield and eucalyptol percentage of 1.35 ± 0.05% and 36.73 ± 1.18%, respectively. After comparison with other techniques in terms of RO yield, separation efficiency, and chemical composition, the HUP-MHD method showed obvious advantages in the separation of RO and oxygenated components (eucalyptol, borneol etc.). In addition, the antioxidant activity (DPPH and ABTS) and antifungal activity of RO from different techniques were measured, the results pointed out that the HUP-MHD method could separate RO with higher biological activity. Therefore, the HUP-MHD has the advantages of short time consumption, high efficiency, and safety in the separation of EO, which provides a theoretical basis for sufficiently separating EO from rosemary and other natural plant and crop materials.