Mesenchymal stromal/stem cells (MSCs) are easily obtained multipotent cells that are widely applied in regenerative medicine. Electrical stimulation (ES) has a promoting effect on bone healing and osteogenic differentiation of MSCs. Direct and alternating currents (AC) are extensively used to promote the osteogenic differentiation of MSCs in vivo and in vitro. However, information on conducting effective differentiation remains scarce. In this paper, we propose a method to optimize ES parameters based on calcium spike patterns of MSCs. Calcium spike frequency decreases as the osteogenic differentiation of MSC progresses. Furthermore, we tested various ES parameters through the real-time monitoring of calcium spike patterns. We efficiently initiated the process of osteogenic differentiation in MSCs by using the optimal parameters of AC, including voltage, signal shapes, frequency, and duty time. This method provides a new approach to optimize osteogenic differentiation and is potentially useful in clinical treatment such as of bone fractures.