Zn(2+) plays vital roles in regulating physiological and pathological processes. A number of diseases are associated with the disruption of intracellular free Zn(2+) homeostasis, and the relationship is still uncovered. Thus, it is important to monitor intracellular free Zn(2+) ions in real time, which is still challenging due to the low content of intracellular free Zn(2+). In this work, we report on the design and synthesis of a new two-photon (2P) fluorescent probe, QZn, based on quinoline derivative for intracellular free Zn(2+). Theoretical calculations were carried out to rationalize the design. The probe displayed a moderate 2P action cross section value of 51 GM at 820 nm and up to 10-fold fluorescence enhancement upon Zn(2+) binding. The detection limit of Zn(2+) was 15.1 pM, which presented a pronounced sensitivity toward Zn(2+) and indicated that QZn would be competent for detecting the low-content intracellular Zn(2+). By using two-photon microscopy, QZn was capable of monitoring the fluctuation of intracellular free zinc ions in real time.