Objective To establish a patch clamp and laser scanning confocal microscope synchronous real-time control system, and apply it in study of membranaceous calcium channel, so as to verify its effects. Methods By adding the patch clamp on the laser scanning confocal microscope, we established a patch clamp and laser scanning confocal microscope synchronous real-time control system using computer autocontrol technique. The system was used to observe the male rat membranaceous calcium channel in vitro, and the observation results were analyzed. Results We successfully established the patch clamp and laser scanning confocal microscope synchronous real-time control system. When the myocardial cells were stimulated, the system could observe the myocardial calcium spark by laser scanning confocal microscope and at the same time record calcium channel current signal by patch-clamp. Quantitative analysis showed that the time intervals between 2 adjacent calcium sparks were (10.055±0.021), (10.079±0.021) and (10.087±0.021) s, which met the stimulus interval for patch-clamp set (10 s). A single calcium spark had a diameter within 2 μm, and it experienced an average period of about 30 ms. It needed an average of 10 ms from the appearance of the spark to its peak concentrations, and it needed an average of 20 ms from its peak concentration to disappearance, which was consistent with the calcium spark theory. Conclusion A patch clamp and laser scanning confocal microscope synchronous real-time control system has been successfully established. The system is capable of performing whole-cell observation recording and determining the membranaceous calcium channel currents and its closing process by patch clamp; at the same time it can synchronously obtain the micro images of the calcium spark with laser scanning confocal microscope, locating changes of calcium ions and helping to understand the internal mechanism of the membrane calcium channels.