The root of Dictamnus dasycarpus Turcz., also known as Cortex Dictamni, is a Chinese herbal medicine that has been commonly used in the treatment of inflammation, microbial infection, cancer, and other diseases in China for thousands of years. Recently, the essential oil of Cortex Dictamni has been widely studied, and a large number of volatile constituents have been discovered. However, the research of the essential oil of Cortex Dictamni in vivo remains unknown, especially the constituents absorbed into blood after oral administration. Hence, a sensitive and rapid method using gas chromatography with mass spectrometry combined with MassHunter software and the National Institute of Standards and Technology 2014 database was used to investigate the absorbed components in rat serum after oral administration of the essential oil of Cortex Dictamni. With the established method, a total of 36 compounds were screened and identified in the essential oil of Cortex Dictamni based on the mass spectrometry data and compound database. Among them, eight compounds, elemol, thymol methyl ether, β-eudesmol, β-cyclocostunolid, guaiazulene, trans-4-hydroxystilbene, ethyl oleate, and monoelaidin, were tentatively characterized in rat serum. This work demonstrated that the established method proved to be a powerful technique for rapid, simple, reliable, and automated identification of bioactive components of herbal medicine.