The purpose of this research is to study the isolation and purification of osthole from Cnidii Fructus extract and to establish a gas chromatographic method for determining the osthole content from various Cnidii Fructus samples. Six solvents were used to extract Cnidii Fructus in order to evaluate their efficiency in extracting osthole. Vacuum fractional distillation and traditional column chromatography were compared to evaluate the efficiency in purifying osthole from Cnidii Fructus extract. Gas chromatographic method was used to determine osthole content in Cnidii Fructus extract. The analytical conditions are the following: nitrogen as the carrier gas, the flow rate of 40 mL/min; the split ratio of 120 : 1. The column used was DBTM-5 (30 m × 0.53 mm I.D., 1.5 μm) equipped with a flame ionization detector (FID). The initial oven temperature was programmed to be at 135 C for 12 minutes. The temperature was then raised to 215°C at a rate of 12°C /min for 20 minutes. The retention time of osthole was 24.1 min. Caffeine anhydrous was used as the internal standard with a retention time of 18.9 minutes. A comparative evaluation showed that aqueous alcoholic solvent was the most efficient solvent among six used in extraction of osthole from a Cnidii Fructus. Vacuum fractional distillation is useful for purifying osthole from Cnidii Fructus extract. It was found that the osthole content of commercial Cnidii Fructus samples varied from 0.05% to 2.14%. And the osthole content of Cnidii Fructus samples collected from Southern China was higher than those collected from Northern China.