Doxorubicin (DOX) is one of the most effective anticancer drugs. However, its therapeutic effectiveness is greatly hampered by its dose limiting and cumulative cardiotoxic side effects. To overcome these limitations, bioconjugates of DOX were studied using bovine serum albumin (BSA) as a carrier to provide passive tumor targeting by the enhanced permeability and retention (EPR) effect. Folic acid, as an active targeting agent, was linked to BSA to increase the selectivity of the conjugate. In the present study, folate-targeted (Folate-BSA-DOX) conjugates were prepared. In the optimization process, we found that 30 mg of folic acid activated esters reacted with BSA at pH 9.8 for 1 h, the yield was maximum. The qualitative analysis of fluorescent experiments revealed that Folate-BSA-DOX can be specifically delivered to Hela cells and that this unique interaction can be blocked by 1 mM free folic acid. More importantly, the enhanced efficiency of uptake of Folate-BSA-DOX by Hela cells was coupled with the increase of the amount of the conjugate, the incubated time and the conjugated ratio of folic acid. Finally, the quantitative data obtained from the flow cytometry further verified the higher targeting and killing ability of Folate-BSA-DOX to folate receptor positive tumor cells than BSA-DOX.