Highly sensitive detection of cancer cells with high signal-to-background ratio (SBR) is still urgently needed. Here, a self-assembling activatable probe (SAAP) based on split aptamers was developed to meet this purpose. The SAAP is formed with quenched fluorescence; only when target cells are present would the split aptamers self-assemble together and thus activate fluorescence by intramolecular and intermolecular fluorescence quenching strategies. As proof of concept, a split aptamer pair stemming from an intact aptamer, ZY11, developed by our lab was selected to construct SAAP. Owing to the design of self-assembly and activation strategy, the SBR of our approach could be raised to ∼40 and achieved a very low detection limit of seven target 7721 cells in 100 μL of binding buffer. Meanwhile, one-step detection of target cells was achieved within 15 min without any washing steps and pretreatment, which shows potential for point-of-care detection. Moreover, we succeeded in the specific recognition of target cells in 50% human serum and mixed cell samples, which indicated this strategy had great advantages in detection in complex biological samples. In addition, dual-signal detection was also successfully implemented, which may be helpful for accurate detection of target cells. Therefore, this rapid, facile, specific, and highly sensitive detection method for cancer cells may provide convenience in cancer research and medical diagnosis.