Impact of steam, hot water blanching and UV-C irradiation as pre-treatments on extraction of oleuropein and related biophenols from olive leaves (OLs), was investigated. Moreover, particle size effect of olive leaves and steam blanching duration were selected as independent variables to optimise steam blanching process in terms of oleuropein content (OC) and antioxidant activity (AC) of ethanolic extracts, by using response surface methodology. Optimum conditions for OC and AC were 10min steam blanching of 20–11 and 3–1mm olive leaf fraction, respectively. Depending on the extraction procedure, at optimum conditions of steaming the results indicate that steam blanching of OL prior to extraction can significantly increase oleuropein yield from 25 to 35 times compared to non-steam blanched sample, whereas the antioxidant activity increased from 4 to 13 times. No significant UV-C effect was observed in OC and AC, while hot water blanched samples showed significantly higher oleuropein yields and antioxidant activity compared to untreated samples.