Background: We investigated whether the condition of the inner surface of hollow fibers affects the blood compatibility of hemodialyzers. Methods: We used scanning probe microscope/atomic force microscopy (SPM/AFM) to investigate the height of the swelling and flexible layers (thickness and softness) on the inner surfaces of the hollow fibers. Next, we tested the blood compatibility between dialyzers comprising a hollow fiber membrane, in which the other dialyzers, except for PVP, were additionally coated using PS membranes coated with other materials. After blood was injected into the dialyzer and plugged, dynamic stimulation was performed by slightly rotating the dialyzer for 4 h, although there was no blood circulation. Results: The vitamin E-coated polysulfone (PS) membrane showed a higher thickness and softness of the flexible layer than the asymmetric cellulose triacetate membrane without polyvinylpyrrolidone (PVP) and the PS membranes with PVP. We found that the dialyzer with vitamin E coating significantly suppressed the decrease in platelets, increase in β-TG, and increase in PF4 compared to those coated with NV polymer. Additionally, as the adsorbed protein on the inner surface, the total protein, fibronectin, and vWF levels were significantly lower in the vitamin E-coated dialyzer. Conclusion: The thickness and softness of the flexible layer of the inner surface of the hollow fiber membrane in vitro affect differences in blood coagulation performance in clinical research. Future clinical trials are required to confirm our results.