Oleuropein was extracted from olive leaves by ultrasonic-assisted method and purified by silica gel column chromatography. The sample was identified as oleuropein by UV, infrared, mass spectroscopy and nuclear magnetic resonance spectroscopy analysis. Oleuropein and total phenolic contents over a year were determined by high-performance liquid chromatography and Folin-Ciocalteu methods. The results showed that 13.52% of pure oleuropein was found with a high purity of 96.54% and purification efficiency of 78.49%. Oleuropein and total phenolic contents of different olive varieties were quite different, but both had similar trends over the year. Five methods, including DPPH (2,2-diphenyl-1-picrylhydrazyl), ABST (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), ferric reducing antioxidant power, total reducing power and nitrite-scavenging ability, were used to evaluate the antioxidant activities of oleuropein, and the IC50 were 34.54 ± 0.14 μg/mL, 18.79 ± 0.82 μg/mL, 75.32 ± 1.83 μg/mL, 13.80 ± 0.68 μg/mL and 1.00 ± 0.08 mg/mL, respectively. PRACTICAL APPLICATIONSThis study provides information on the changes in contents including oleuropein and total phenolics in a year to help understand the relationship between content of oleuropein and various influencing factors. Currently, the metabolic pathways of oleuropein are not clear. These results provide information for the study of the metabolism of phenolic compounds in olive and promote research at the molecular level. Oleuropein occupies a large proportion of total polyphenols in olive and can be to be purified by silica gel column chromatography with high purification efficiency for industrial production and laboratory production. In addition, the comprehensive evaluation of activity in vitro shows that oleuropein is more useful than BHT (2,6-di-tert-butyl-4-methylphenol) for the application in food and human health.