Autophagy is an adaptive catabolic process functioning to promote cell survival in the event of inappropriate living conditions such as nutrient shortage and to cope with diverse cytotoxic insults. It is regarded as one of the key survival mechanisms of living organisms. Cells undergo autophagy to accomplish the lysosomal digestion of intracellular materials including damaged proteins, organelles, and foreign bodies, in a bulk, non-selective or a cargo-specific manner. Studies in the past decades have shed light on the association of autophagy pathways with various diseases and also highlighted the therapeutic value of autophagy modulation. Hence, it is crucial to develop effective approaches for monitoring intracellular autophagy dynamics, as a comprehensive account of methodology establishment is far from complete. In this review, we aim to provide an overview of the major current fluorescence-based techniques utilized for visualizing, sensing or measuring autophagic activities in cells or tissues, which are categorized firstly by targets detected and further by the types of fluorescence tools. We will mainly focus on the working mechanisms of these techniques, put emphasis on the insight into their roles in biomedical science and provide perspectives on the challenges and future opportunities in this field.