Tyrosinase (TYR) plays an important role in melanin formation and is regarded as the biomarker for melanoma cancer. Highly sensitive and selective detection of TYR activity in live cells may enhance the comprehension on the mechanism of TYR in living organisms and promote the prognosis and precise diagnosis of TYR related diseases. Herein, an “off-on” near-infrared fluorescent chemosensor EQR-TYR was developed that could detect TYR with high anti-interference ability. The evaluation of TYR activity was established in terms of the relationship between fluorescent intensity of EQR-TYR and TYR activity. Importantly, EQR-TYR showed various advantages, such as good water-solubility, low detection limit of TYR (0.035 U/mL), remarkably large Stokes shift (130 nm) and fast response (1 h). The application of EQR-TYR was successfully demonstrated by monitoring different TYR expressions in living cells and xenograft tumor mouse model, indicating its good biocompatibility. We anticipate that the novel chemosensor will provide an alternative for monitoring TYR activity in complex biological systems.