Graphitic-C3N4 quantum dots (g-C3N4 QDs) have been widely studied because of their excellent water solubility, high quantum yield and low toxicity. Herein, we propose a new pathway for the preparation of g-C3N4 QDs by a fast one-step method involving the disintegration of bulk g-C3N4 in water at high temperature. This method is facile and rapid in comparison with other procedures. The prepared g-C3N4 QDs have a size of about 3 nm and exhibit strong blue fluorescence under a 365 nm UV lamp. Further experiments demonstrate that the g-C3N4 QDs prepared by this method are suitable for application in two-photon imaging, suggesting that this one-step method may provide an innovative way for the preparation of environmentally friendly probes for two-photon imaging.