A promising, safe, and economic fluorescent probe, g-C3N4 single-layered QDs, is introduced for two-photon fluorescence imaging of the cellular nucleus for the first time. The large two-photon absorption cross section, the high photostability, good biocompatibility and non-toxicity, negligible photothermal effect, and specific interaction with DNA render the single-layered g-C3N4 QDs as a promising candidate for in vivo and in vitro two-photon fluorescence imaging and further biomedical applications.