Traditional studies on the relationship between hyperglycemia and heart diseases generally focused on the impact of chronic and long-term effect of diabetes on cardiac functions. Most of the methods were culturing myocardial cells and giving outside stimulations. However, recent studies show that acute hyperglycemia might play a significant role in spontaneous cardiac electrophysiology. In this research we applied microelectrode arrays (MEA) to record the spontaneous sinoatrial node field potentials of C57/BL6J mice and analyzed the effects of different glucose concentrations in time domain and frequency domain by using statistical method, vector maps and fast Fourier transform (FFT). Meanwhile, we studied the effects of insulin interference in the experimental process. When the concentration of the glucose solution was greater than 40 mmol/L, the spontaneous sinoatrial node field potential changed markedly. In the time domain, the amplitude decreased rapidly and the conductive characteristics were disordered. In the frequency domain, the two spectrum peaks decreased rapidly. These changes were irreversible. However, insulin preconditioning could inhibit the impact of high glucose.