The content of piperine in the flower,young and ripen fruit of pepper were investigated by RP-HPLC with Diamonsil column (C18,5μm ,150 mm×4. 6 mm) at room temperature and 343 nm. The mobile phase was water-methanol (gradient elution) with flow rate of 1mL/min. The piperine was used as the standard. The result showed that the linear range was 3-150 μg/mL ,and correlation coefficient was 0.9994.The limit of detection was 1.2 μg/mL. Retention time was 12.3 min. Contents of piperine was 4.153 mg/mL in young fruit and 27.2 mg/mL in the ripen fruit. Their relative standard deviations were 1.9% and 0.95%,respectively.No piperine was found in flowers. The average recovery of green pepper was 97.06% while ripen pepper was 96.66%. The relative standard deviations were 0.95% and 1.21% ,respectively. These results concluded that the method was simple,accurate ,and can be used for determining the content of pipperine in the flower,young and ripen fruit of pepper.