Targeting antitumor drugs to specific tissues or cells has attracted considerable interest in the recent tumor therapy because it can reduce the side effects in the body and the treated drug dosage through homing drug to the desired tissues. To utilize the high affinity of biotin to avidin as a drug carrier for doxorubicin (DOX), biotinylated targeting material (biotin-polyethylene glycol (PEG)-folic acid, BPF) and biotinylated drug with a bioreducible linker (biotin-SS-PEG-DOX, BSPD) were designed in this study. The in vitro cellular uptake of the avidin/biotinylated drug complexes showed that the complexes with BPF were internalized dose-dependently into HeLa cells, while their uptake was not detected in the absence of BPF. Although the anti-tumor activity of the drug complexes with BPF and BSPD were similar to that of free DOX in cervical cancer cells, the cytotoxic effect of DOX was significantly reduced in normal cells. Their targeting antitumor effect revealed the significant inhibition of the increasing tumor size in HeLa-xenograft mouse model. These results show that the strong complexing between avidin and biotin acted as a targeting moiety and the antitumor drug is an adaptable tool in the fields of tumor therapy.