The development of simple and versatile approaches for the fabrication of DNA-based composite nanomaterials, endowed with defined morphologies and specific functionalities, is of paramount importance for various applications. Herein, we report a simple approach for the synthesis of multifunctional copper-DNA nanoflowers (Cu-DNF) that exclusively consist of rolling circle polymerized nanoflowers (DNF) and in situ synthesized concatemeric fluorescence copper nanoparticles. Through meticulous regulation of the assembly process, it is possible to generate Cu-DNF with precise sizes and stable fluorescence properties. The obtained Cu-DNF possesses robust biostability to resist degradation by nuclease, presumably resulting from the dense structure of the Cu-DNF. The Cu-DNF were also encoded with polyvalent tandem CD63 aptamer sequences, which enhanced their binding affinity and internalization efficiency into tumor cells. We demonstrate that the multifunctional Cu-DNF can efficiently internalize tumor cells for tracking and imaging analysis of intracellular microRNA. This approach may be beneficial for creating multifunctional DNA-based composite nanomaterials for various technological applications.