Doxorubicin (DOX) is one of the most effective anti-cancer drugs, but its therapeutic efficacy is greatly hampered by its non-specific delivery to the target tissue and the resultant cumulative cardiotoxicity and nephrotoxicity. In order to overcome this limitation, we prepared a folate-bovine serum albumin (BSA)-cis-aconitic anhydride-doxorubicin prodrug, denoted by FA-BSA-CAD. A tumor-targeting agent, folic acid, was linked to BSA to increase the selective targeting ability of the conjugate. BSA provided a large number of reactive sites for multivalent coupling of bioactive molecules and improved the water-solubility of the prodrug. DOX is attached to the BSA via a pH-sensitive linker, cis-aconitic anhydride, which hydrolyzes in the acidic lysosomal environment to allow pH-responsive release of DOX. The in vitro results demonstrate a pH-responsive drug release under different pH conditions. Furthermore, the targeting ability and therapeutic efficacy of the prodrug were assessed both in vitro and in vivo. The results demonstrate that FA-BSA-CAD prodrug selectively targeted tumor cells and tissue, with associated reduction in non-specific toxicity to the normal cells. More importantly, the therapeutic efficacy of the prodrug for FA-positive tumors increased compared to the non-conjuagted DOX.