Pancreatic β-cells release insulin in a Ca²⁺-dependent pulsatile manner to control blood glucose levels. Spatiotemporal kinetics and function of Ca²⁺ signaling in β-cells have been studied by experiments using in vitro and ex vivo preparations. However, in vivo analysis remains challenging. While β-cell activities in vivo are under the influence of the autonomic nervous system, hormones and other bioactive substances, Ca²⁺ activities of β-cells 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 genetically encoded ratiometric Ca²⁺ indicator, 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 clarify the basis for a pulsatile insulin secretion. Further studies using current method is expected to gain deeper insight in insulin secretion mechanism and the etiology of diabetes.