BackgroundAutophagy is a critical self-eating pathway involved in numerous physiological and pathological processes. Lysosomal degradation of dysfunctional organelles and invading microorganisms is central to the autophagy mechanism and essential for combating disease-related conditions. Therefore, monitoring fluctuations in the lysosomal microenvironment is vital for tracking the dynamic process of autophagy. Although much effort has been put into designing probes for measuring lysosomal viscosity or pH separately, there is a need to validate the concurrent imaging of the two elements to enhance the understanding of the dynamic progression of autophagy.MethodsProbeHFIwas synthesized in three steps and was developed to visualize changes in viscosity and pH within lysosomes for real-time autophagy tracking. Then, the spectrometric determination was carried out. Next, the probe was applied to image autophagy in cells under nutrient-deprivation or external stress. Additionally, the performance ofHFIto monitor autophagy was employed to evaluate acetaminophen-induced liver injury.ResultsWe constructed a ratiometric dual-responsive probe,HFI, with a large Stokes shift over 200 nm, dual-wavelength emission, and small background interference. The ratiometric fluorescent signal (R =I610/I460) ofHFIhad an excellent correlation with both viscosity and pH. More importantly, high viscosity and low pH had a synergistic promotion effect on the emission intensity ofHFI, which enabled it to specially lit lysosomes without disturbing the inherent microenvironment. We then successfully usedHFIto monitor intracellular autophagy induced by starvation or drugs in real-time. Interestingly,HFIalso enabled us to visualize the occurrence of autophagy in the liver tissue of a DILI model, as well as the reversible effect of hepatoprotective drugs on this event.ConclusionsIn this study, we developed the first ratiometric dual-responsive fluorescent probe,HFI, for real-time revealing autophagic details. It could image lysosomes with minimal perturbation to their inherent pH, allowing us to track changes in lysosomal viscosity and pH in living cells. Ultimately,HFIhas great potential to serve as a useful indicator for autophagic changes in viscosity and pH in complex biological samples and can also be used to assess drug safety.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40824-023-00409-3.