Autophagy (“self-eating”) is an evolutionarily conserved survival mechanism in living organisms during which intracellular components (‘cargo’) are identified and delivered to lysosomes for degradation.1 Basal autophagy plays a vital role in maintaining cellular homeostasis, such as keeping the amino acid pool recycling in the event of nutrient deprivation, clearing the toxic accumulation of damaged or unwanted components, and suppression of cell proliferation.2 Dysregulation of autophagy have been found to associate with diseases ranging from neurodegeneration, early stage of cancers, cardiovascular disease to infectious disease, with different stages of autophagy being impaired.3 Research efforts have identified multiple molecular targets to rectify autophagy with the promise for therapeutic intervention.4 Modulators that enhance or inhibit autophagic activities have largely contributed to mechanistic understanding of autophagy and their implications in diseases. As autophagy is a multi-step process, it is important to identify and assess which stage of autophagy is affected by a modulator, to quantify the extent of the regulation to avoid undesirable side effect and to draw a clear mechanistic picture. However, tools that allow real-time monitoring of the dynamics of autophagy, especially with quantitative readout, are still scarce and highly desirable. Conventional methods involving the expression of fluorescent protein tagged autophagy markers require tedious and complexed transfection procedures and fail to be applicable in clinical samples. Here, by incorporating the autophagy-targeting chimera, we design and synthesize the first fluorescent chemical probe that is highly specific to autophagy. Also, the introduction of a pH-sensitive fluorophore into the molecular design enables the discrimination between autophagosomes and acidic autolysosomes. This tailor-made probe, named AUTag, has been proven to be applicable in live cells and in vivo under conditions that induce/inhibit autophagy, which was validated with standard autophagy methods. Also, by virtue of AUTag, protocols based on flow cytometry or plate reader for quantitative analysis of cellular autophagic flux have been established. With a series of advantages, such as fast cell penetration, ease of operation and high specificity, the new autophagy sensor was further utilized to study the role of autophagy during embryotic development in zebrafishes. In general, the utilization of AUTag not only provides a robust tool for autophagy research but also facilitates the applications for drug screening, understanding disease mechanisms, as well as studying fundamental biological processes.