The major flavonoids in rat serum after oral administration of Dalbergia odorifera extract were analyzed qualitatively and quantitatively by high performance liquid chromatography (HPLC) and its coupling to mass spectrometry (HPLC-MS). Utilizing HPLC-MS technique, 18 flavonoids including five isoflavones, four isoflavanones, four neoflavones, two flavanones, two chalcones, one isoflavanonol were identified in free form in serum sample based on comparison with the authentic standards. Furthermore, the amounts of the four prominent flavonoids, (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone, vestitone, formononetin and sativanone were determined in serum by HPLC-UV with internal standard method. The method was validated and utilized in pharmacokinetic studies of these four analytes. This is the first report on identification and determination of the major flavonoids in rat serum after oral administration of D. odorifera extract and the results provided a firm basis for clarifying the pharmacological effect of D. odorifera and evaluating the clinical applications of this medicinal herb.