A novel quinazolinone-based fluorescent dye, 6-dimethylamino-2-(quinolin-2-yl)- quinazolin-4(3H)-one (DMAQQ), has been designed and synthesized. This quinazolinone-based dye exhibited good efficient solvatochromic fluorescence with appreciated fluorescence quantum yield in different organic solvents (0.57–0.78) and remarkable two-photon fluorescence. The theoretical calculation showed that it was derived from charge transfer (CT) character and intramolecular hydrogen bond. Further investigation on the interaction with anions also showed that the fluorescence intensity of this dye decreased and quenched through the destruction of intramolecular hydrogen bond by the addition of hydrogen bond strong acceptor. As a good bioimaging candidate, DMAQQ showed excellent biocompatibility. More importantly, DMAQQ exhibited blue and green one-photon fluorescence emission in HeLa cells with the Pearson's coefficient 0.39, which suggested possible different local polarity in HeLa cells. And the DMAQQ also showed bright green fluorescence in HeLa cells, which excited by two-photon, offering a new dye platform for high spatial resolution bio-imaging application.