Development of smart DNA nanostructures is of great value in cancer studies. Here, by integrating rolling circle amplification (RCA) into split aptamer design, a novel strategy of polyvalent and thermosensitive DNA nanoensembles was first proposed for cancer cell detection and manipulation. In this strategy, a long nanosolo ssDNA with repeated Split-b and Poly T regions was generated through RCA. Split-b supplied polyvalent binding sites while Poly T supported signal output by hybridizing with fluorophore-labeled poly A. After addition of Split-a, nanoensembles formed on the cell surface due to target-induced assembly of Split-a/Split-b from the free state to the recognition structure, and on the basis of the thermosensitivity of split aptamer, nanoensembles were controlled reversibly by changing temperatures. As proof of concept, split ZY11 against SMMC-7721 cancer was used to construct nanoensembles. Compared with monovalent split aptamer, nanoensembles were demonstrated to have a much stronger interaction with target cells, thus realizing an ∼2.8-time increase in signal-to-background ratio (SBR). Moreover, nanoensembles extended the tolerance range of target binding from 4 °C to room temperature and speeded recognition thus achieving almost 50% binding in 1 min. Then, nanoensembles were successfully applied to detect 7721 cells in serum and mixed cell samples. By utilizing microplate well surface as the model, temperature-controlled catch/release of target cells was also realized with nanoensembles, even under unfriendly conditions for monovalent split aptamer. The RCA-mediated aptameric nanoensembles strategy not only solved the problem of split aptamer in inefficient binding but also paved a brand new way for developing polyvalent and intelligent nanomaterials.