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Viscosity is a key parameter of mitochondria that related to several diseases. To design a probe acting as both the mitochondria-targeted viscosity sensor and photosensitizer is meaningful for diagnosis and therapies. Herein, we report a ratiometric viscosity sensor with dual-emission mediated by excitation energy transfer (EET) effect, which can detect the mitochondrial viscosity and serve as an efficient photosensitizer to eradicate cancer cells. The probe comprises of a blue-emitting imidazole moiety as the energy donor and a green-emitting boron dipyrromethene (BODIPY) core as the energy acceptor, respectively. Via viscosity-modulated EET effect, the dual-emissive probe can sensitively detect the medium viscosity in a ratiometric model. Directed by imidazole as the targeting factor, the probe can specifically accumulate in mitochondria and ratiometrically determine the intracellular viscosity increase induced by Nystatin treatment. Furthermore, it also exhibits light-triggered reactive oxygen species (ROS) generation including singlet oxygen and superoxide radical, which can efficiently kill the cancer cells under white light illumination. Thus, this work provides a versatile and promising candidate for disease diagnosis and phototherapy. [Display omitted] • Excitation energy transfer (EET) conjugated molecule shows viscosity-mediated dual-emission fluorescence property. • The fluorophore can specially target mitochondria and ratiometrically reveal the viscosity change. • Viscosity sensor exhibits light-triggered ROS generation for efficient photokilling on dysfunctional cells. [ABSTRACT FROM AUTHOR]