Objective To investigate the effect of fluid shear stress (FSS) on VWF-A1A2A3-mediated expression of platelet surface P-selectin. Methods Using a parallel plate flow chamber system and mouse anti-human CD62P: FITC as the indicator of P-selectin expression, the alteration of platelet P-selectin expression level with increasing exposure time under different FSS conditions (0, 1, 2 Pa) specifically mediated by VWF-A1A2A3 was observed and analyzed by fluorescence microscope to obtain the activation characteristics. Results FSS triggered platelet activation and P-selectin expression. The activation ratio of platelet was positively regulated by FSS and their exposure time, reaching the maximum value, 9.42% and 14.59% under FSS of 1 Pa and 2 Pa, respectively. The level of P-selectin expression exhibited two-phase tendency with the shear stress-exposure time increasing, uplifted at first, then decreased, with the best action time at 7.5 min. The fluorescence peak intensity increased when FSS was enhanced. Conclusions The level of platelet P-selectin expression is co-regulated by VWF-A1A2A3 and FSS, and is closely related to force-signaling exposure time.