A reversed-phase high performance liquid chromatographic (RP-HPLC) method for the determination of the tetramethylpyrazine(TMP) in Chuanxiong extract, the animal(mouse) serum and cerebrospinal fluid has been developed. The TMP was separated on an ODS column Zorbax SB-C18(4.6 mm i.d. x 250 mm, 5 microns) at room temperature and detected by using UV detector at 270 nm. The mobile phase was methanol-water (50:50, V/V) containing 0.2 mmol/L of NH4H2PO4 flowing at a rate of 0.8 mL/min and 20 microL samples were injected. The detection limit of TMP was 1 mg/L and the calibration curve is linear between 5 and 500 mg/L with a correlation coefficient (r) of 0.999. The recovery of TMP ranged 98%-103%. The extract of Chuanxiong and pretreated serum and cerebrospinal fluid sample are stable for a week at room temperature.