Viscoelastic property and morphology of fibrin, which is caused by the enzymatic action of thrombin on fibrinogen, was studied quantitatively as a function of antithrombin III (AT) and heparin concentration by adding fibrinogen, heparin, AT, and thrombin, sequentially. A quartz crystal microbalance with impedance analysis (QCM-Z) was used to detect the change of viscoelastic properties as well as the thickness of adsorbed layer during fibrin formation process, while AFM was used to characterize the surface morphology of fibrin layer under the influence of two known anticoagulants. By the addition of fibrinogen initially, a rigid and thin fibrinogen layer with rather smooth surface morphology was formed on the substrate. Then, the addition of thrombin in the absence of AT and heparin, resulted in viscous and thick fibrin gel with textured surface morphology. As an anticoagulant, AT was added before the injection of thrombin, but AT in the absence of heparin showed only marginal effects at higher concentration of AT than that of thrombin. On the contrary, the thick and viscous fibrin layer was altered gradually to more fibrinogen-like layer as the heparin concentration increases at low concentrations of AT, demonstrating the powerful anticoagulant effect by heparin/AT complex. Interestingly, heparin alone without AT was also effective in preventing fibrin formation, influencing the viscoelastic property and morphology of fibrin.