Objective To investigate the shear rate and VWF-A1-mediated platelets calcium response. Methods Flou-4 AM was used as the fluorescent indicator of intracellular calcium, and the intracellular calcium concentrations of adherent platelets on VWF-A1 with different concentrations at different shear rates were detected by parallel plate flow chamber system combined with fluorescence microscope. The effect of shear stress and chemical signaling on intensity and speed of calcium response was also analyzed. Results VWF-A1 specifically mediated platelet adhesion and calcium response under flow. The shear rate and molecular concentrations had positive regulation of platelets activation ratio and negative regulation of delay time of calcium response. Compared with static conditions, the activation ratio was increased by 37 times at shear rate of 500 s-1. When the concentration of VWF-A1 was increased from 60 mg/L to 240 mg/L, the delay time of calcium response was shortened by 10 s. Conclusions The shear rate could modulate the calcium response of platelets in cooperation with VWF-A1. The result of this study will be helpful to deepen the understanding about the process of hemostatic reaction of platelet coagulation in bloodstream.