Gas chromatography (GC)-mass spectrometry is a powerful tool which is used in the analysis of volatile components in food and herbal medicine. Euphorbia fischeriana Steud., a traditional Chinese herb, has been used for thousands of years in China. Recently, scientists are exploring more on the pharmacological action of E. fischeriana. However, so far, there is no information regarding the volatile constituents of E. fischeriana. Therefore, in this study, we aimed to investigate and characterize the global chemical ingredients of volatile oil of E. fischeriana under in vitro and in vivo conditions. In this study, an accurate and rapid GC combined with the mass spectrometric method and MassHunter tool were employed to identify the comprehensive constituents and explored the absorbed components in rat serum after oral administration of E. fischeriana. According to our results, a total of 28 compounds were identified via in vitro analysis by matching with the NIST database. Of them, the following 14 compounds were tentatively characterized in the serum samples: (−)-alpha-cedrene; (+/-)-sesquithuriferone; cedrol; acorenone; neocembrene; geranylgeraniol; abietatriene; abieta-7,13-diene; cembrenol; sclareol; ethyl linoleate; isopimara-7,15-dien-3-one; abietadienal; and abietinol. Most of the compounds identified were diterpenes and sesquiterpenes. These results might be useful in various pharmacological and pharmacodynamic research, and the established method could offer valuable chemical information for bioactive volatile components characterization.