The separation of carrier-free90Y from90Sr by solvent extraction with di-(2-ethylhexyl) phosphoric acid (HDEHP) in dodecane has been investigated in this study. The extraction equilibrium time, the effect of temperature, concentration of extractant, and hydrochloric acid concentration on this extraction system were examined. Based on the studies, the chemical procedure for the separation of carrier-free90Y from its parent90Sr have been established. The chemical yield of90Y is about 80%. Meanwhile, the90Sr impurity is less than 1×10−6% and the trace of extractant presented in the final product can be removed by fumed with HNO3. Consequently, the preparation of millicurie of90Y is quite satisfactory for the adsorption in microsphere resin for radiotherapy applications.