In order to detect the content of aucubin in samara of Eucommia ulmoides by high performance liquid chromatography(HPLC), Eucommia ulmoides samara was taken as raw materials, 85 percent ethanol was used at the condition of temperature 50 ℃, liquid ratio 1∶20, extraction for 2 times and 30 min for each time to extract aucubin till there is no alcohol taste in the concentrated extracts. The purpose of this research is to develop a set of useful HPLC analysis approach of Eucommia ulmoides aucubin from the detection wave length, mobile phase, flow velocity and temperature. The results showed that optimal separation was achieved on a C18 (150 mm×4.6 mm, 5 μm) column, whenits mobile phase ranged from 1 to 10 percent acetonitrile, gradient elution, and its flow rate was 0.8 mL/min, its column temperature was 20 ℃ and its wavelength was set at 208 nm. The calibration curve was linear within the range of 0.30~1.50 μg, R2=0.999 6, the average recovery for aucubin was 84.3%, RSD was 1.97%(n=5). This analysis method could be used to detect the content of Eucommia ulmoides aucubin with simple samples, stability, good repeatability and high precision.