Abstract: In order to explore the optimal concentration and the time of Fluo-3 AM loading adherent Rat Myocardial Microvascular Endothelial Cells (RMMECs). Fluo-3AM were diluted to 1, 2, 3, 4, 5 μM/L and each concentration of the probe was mounted cells for 5, 10, 15, 20 min. Laser Scanning Confocal Microscope (LSCM) was used to detect intracellular fluorescence signal. To observe the loading effect according to the fluorescence intensity, position and changes of cells morphology after loading probe. The results showed that: the fluorescence signal did not appear after loading 5 to 20 min by 1 to 3 μM/L Fluo-3AM, the fluorescence signal began to appear after loading 5 to 10 min by 4μM/L Fluo-3AM, Clear and uniform distributed fluorescence signal could be seen after loading 15 min by 5μM/L Fluo-3AM. The effect of Fluo-3AM loading adherent RMMECs was mainly affected by the concentration of probe and loading time.