Pancreatic β-cells release insulin in a Ca²⁺-dependent pulsatile manner to control blood glucose levels. Although the Ca²⁺ signaling mechanism in β-cells has been extensively studied using in vitro and ex vivo preparations, its analysis in living animals remains challenging. Therefore, while β-cell activities in vivo are under the influence of the autonomic nervous system, various hormones and other bioactive substances, Ca²⁺ responses of β-cells under physiological conditions have not been clarified. We here report a method to monitor and analyze in vivo β-cell Ca²⁺ activities 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 in living animals using the new method is expected to help elucidate the mechanism of insulin secretion and the etiology of diabetes.