Interplay between calcium ions (Ca(2+)) and reactive oxygen species (ROS) delicately controls diverse pathophysiological functions of vascular smooth muscle cells (VSMCs). However, details of the Ca(2+) and ROS signaling network have been hindered by the absence of a method for dual measurement of Ca(2+) and ROS. Here, a real-time monitoring system for Ca(2+) and ROS was established using a genetically encoded hydrogen peroxide indicator, HyPer, and a ratiometric Ca(2+) indicator, fura-2. For the simultaneous detection of fura-2 and HyPer signals, 540 nm emission filter and 500 nm~ dichroic beamsplitter were combined with conventional exciters. The wide excitation spectrum of HyPer resulted in marginal cross-contamination with fura-2 signal. However, physiological Ca(2+) transient and hydrogen peroxide were practically measurable in HyPer-expressing, fura-2-loaded VSMCs. Indeed, distinct Ca(2+) and ROS signals could be successfully detected in serotonin-stimulated VSMCs. The system established in this study is applicable to studies of crosstalk between Ca(2+) and ROS.