Abstract: Chemotherapeutic drugs generally lack specificity, and so the development of a carrier that can actively target delivery of chemotherapy drugs without immunogenicity to organisms has attracted increasing attention. In this work, a multivalent aptamer (Multi-Apt) was constructed by hybridizing a long single-stranded DNA (ssDNA) with tandem repeated sequences synthesized by rolling circle amplification (RCA) with several ssDNA encoding aptamer AS1411 sequences. The double-helix structure was used to load the anti-tumor drug doxorubicin (Dox) for targeted treatment of B16 cells. The binding ratio of RCA product to ssDNA was optimized by agarose gel electrophoresis, and the loading and release of Dox were explored using a microplate reader. The targeting and growth inhibition of Multi-Apt-Dox on B16 cells were investigated by fluorescence microscopy, flow cytometry, microplate reader, CCK-8 assay and wound healing assay. The results showed that the optimal molar ratio of RCA product to ssDNA was 1:50. Fluorescence microscopic pictures, flow cytometry analysis and microplate reader experimental results showed that each Multi-Apt could load approximately 200 Dox molecules, and the affinity of Multi-Apt for B16 cells was 46-fold higher than that of free AS1411. Cell experimental results demonstrated that Multi-Apt induced selective cytotoxicity after intracellular degradation and drug release, thereby greatly reducing the adverse reactions of Dox to normal cells and providing a new strategy for targeted drug delivery in tumor treatment.