Pancreatic β-cells release insulin to control glycemia in an intracellular Ca²⁺-dependent manner. Spatiotemporal dynamics and the function of Ca²⁺ signaling in β-cells have been studied by experiments using in vitro and ex vivo preparations. However, in vivo study remains challenging. Unlike the case of in vitro/ex vivo preparations, β-cells in vivo are under the influence of the autonomic nervous system, hormones, and other bioactive substances. Thus, β-cell Ca²⁺ activities under physiological conditions have not been clarified. We here report a method to analyze in vivo β-cell Ca²⁺ signals using a transgenic mouse line expressing a ratiometric Ca²⁺ indicator protein, YC-Nano50. Using the method, we visualized β-cell Ca²⁺ signals in laparotomized mice under anesthesia, and observed synchronized Ca²⁺ oscillations in β-cells within individual islets. Furthermore, we succeeded in monitoring Ca²⁺ activities in multiple islets simultaneously, which may provide unprecedented clues to understand the regulation mechanism of intravital insulin dynamics. Further studies in living animals using the new method are expected to help elucidate the mechanism of insulin secretion and the etiology of diabetes.