Abstract: Seasonal variation of oleuropein content in olive leaves was investigated, and the influences of preparation and extraction factors to the yield were discussed. HPLC was used to determine the content of oleuropein. Results showed: content of oleuropein in a year has two peak periods,namely Feb. and May (among them: Feb.＞May), and two trough periods,namely Nov. and April. Huge differences existed among different breads. Keladi olive leaves have the highest oleuropein content of 17.2%, in contrary, Peidaling olive leaves have the lowest oleuropein content of 1.65%. The content of oleuropein of 20 years-old trees is much higher than that of 3 years old. Ultrasonic extraction is better than heat extraction. Natural drying of leaves superior to heat drying to obtain higher yield of oleuropein.