Insulin secretion is a complex and highly regulated process. Although much progress has been made in understanding the cellular mechanisms of insulin secretion and regulation, it remains unclear how conclusions from these studies apply to living animals. That few studies have been done to address these issues is largely due to the lack of suitable tools in detecting secretory events at high spatial and temporal resolution in vivo. When combined with genetically encoded biosensor, optical imaging is a powerful tool for visualization of molecular events in vivo. In this study, we generated a DNA construct encoding a secretory granule resident protein that is linked with two spectrally separate fluorescent proteins, a highly pH-sensitive green pHluorin on the intra-granular side and a red mCherry in the cytosol. Upon exocytosis of secretory granules, the dim pHluorin inside the acidic secretory granules became highly fluorescent outside the cells at neutral pH, while mCherry fluorescence remained constant in the process, thus allowing ratiometric quantification of insulin secretory events. Furthermore, mCherry fluorescence enabled tracking the movement of secretory granules in living cells. We validated this approach in insulin-secreting cells, and generated a transgenic mouse line expressing the optical sensor specifically in pancreatic β-cells. The transgenic mice will be a useful tool for future investigations of molecular mechanism of insulin secretion in vitro and in vivo.