The DNA nanosphere (DNS) is constructed through the assembly of multifunctional Y-scaffold DNA monomers, which encompass aptamers, RNA interferences, and photodynamic therapy agents. The DNS nanoplatform facilitates efficient cellular uptake via aptamer-mediated delivery. The RNA interferences inhibit the microRNA-21 and microRNA-155, thereby enabling precise imaging guidance for photodynamic therapy, ultimately leading to apoptosis in MCF-7 cells. The platform allows for real-time monitoring of the therapeutic process, presenting a more comprehensive approach to cancer treatment. The results of in vitro and in vivo studies suggest the potential of this multifunctional DNS platform as a promising approach for cancer therapy that can be tailored to individual patients based on their specific cancer type.