Cuprous ions (Cu+) are essential for various biological activities. However, it is still a great challenge to detect Cu+ efficiently in cells in situ. As emerging 0‐D carbon photoluminescence nanomaterials, carbon dots (CDs) exhibit non‐toxicity and ultra stability, which shows great advantages in biosensing and bioimaging. In this work, CDs are prepared by a one‐step microwave‐assisted hydrothermal method. To enhance the selectivity and efficiency of the CDs‐based sensor, the NS4‐CDs are synthesized by surface modification of CDs using Cu+ chelating molecules. The NS4‐CDs, that carry specific detection molecules, exhibit remarkable selectivity and high sensitivity with an extraordinarily low detection limit (26 nmol L−1). It is noteworthy that the detection response is extremely fast, which only takes 20 s. The PL response mechanism of NS4‐CDs is also thoroughly investigated. In addition, the NS4‐CDs possess great biocompatibility and non‐cytotoxicity, which is vital for advanced cellular imaging applications. Finally, the NS4‐CDs are successfully applied to Cu+ detection in cells in situ. This work paves a new path for the design and efficient construction of CDs‐based biosensors for ion detection in cells, which expands the application area of carbon nanomaterials.