Platelets are rapidly activated by activators and produce a large number of platelet microparticles (PMPs) with high coagulation activity, resulting in coagulation dysfunction. However, the generation mechanism of PMPs is still not clear. Hopping probe ion conductance microscopy (HPICM) has special technical advantages in non-contact, real-time, high-resolution imaging of living cells under physiological conditions. Using HPICM, this study monitored the processes of platelet activation and generation of PMPs in real time in the presence of calcium ionophore A23187 and cytochalasin D (CD), respectively. The results proved that the intracellular calcium concentration and the cytoskeletal proteins played important roles in the platelet activation and the generation of PMPs. Compared with the low density spread shape platelets (LDSS), the high density bubble shape platelets (HDBS) were more sensitive to the calcium ionophore A23187 and cytochalasin D. This research has a guiding significance for the further study on the relationship between platelet activation and coagulation function using HPICM.