Carbon dots (CDs) are emerging as powerful tools for biosensing and bioimaging because of their intrinsic properties such as abundant precursors, facile synthesis, high biocompatibility, low cost, and particularly robust tunability and stability. In this work, a new type of CDs was prepared from m-phenylenediamine and folic acid by hydrothermal method. Interestingly, the as-prepared CDs show blue emission in non-hydrogen-bonding solution, whereas robust green emission in hydrogen-bonding solution. Based on this phenomenon, a novel fluorescence sensing mechanism named as hydrogen-bonding-induced emission (HBIE) was proposed. The HBIE-CDs have large Stokes shift (141 nm) in water, good biocompatibility, and ultrasmall size, which facilitates their translocation into living cells. Very importantly, the as-prepared HBIE-CDs show strong affinity toward nucleic acid without interference from other biological species. After binding with DNA/RNA through hydrogen bond, as high as 6-fold green fluorescence enhancement of HBIE-CDs was observed. Since the nucleus is rich in DNA/RNA, these HBIE-CDs were successfully used for rapid and, especially, wash-free subcellular in situ imaging of the nucleus in living cells in a fluorescence turn on mode, which has a great practicability to be used for nucleus imaging in bioanalytical studies and clinical applications.