Our understanding of cellular signaling is critically dependent on our ability to visualize and quantify specific signaling events with high spatial and temporal resolution in the cellular context. Over the past decade or so, biosensors based on fluorescent proteins and fluorescence resonance energy transfer (FRET) have emerged as one major class of fluorescent probes that are capable of tracking a variety of cellular signaling events, such as second messenger dynamics and enzyme activation/activity, in time and space. Here we review recent advances in the development of such biosensors and some biological insights revealed by these biosensors in living cells, tissue, and organisms.