Visualizing the function of pancreatic β-cells in vivo has been a long-sought goal for β-cell researchers. Unlike imaging of β-cells in mammalian species with conventional positron emission tomography and single-photon emission computed tomography, which only provides limited spatial-temporal resolution, transparent zebrafish embryos are a unique model that allows high-resolution fluorescent imaging of β-cells in their native physiological microenvironment in vivo. Here, we detail a protocol for real-time visualization of individual β-cell function in vivo in a non-invasive manner, through combination of a novel transgenic zebrafish reporter line Tg (ins:Rcamp1.07) with both a commercial spinning-disc confocal microscope and an in-house developed super-resolution microscope (2P3A-DSLM). The protocol described here allows for the longitudinal monitoring of dynamic calcium activities from heterogeneous β-cells in early developing zebrafish embryos and is readily adaptable for use in imaging other important processes in islet biology, as well as screening new compounds that can promote β-cell function or maturation using a living whole organism system.