Anticoagulation factor I (ACF I) from the venom of Agki~strodon acutus is a binding protein to activated coagulation factor X (FXa) and possesses marked anticoagulant activity. Single ACF I molecule has been successfully imaged in air by tapping mode atomic force microscopy (AFM) with high resolution using glutaraldehyde as a coupling agent. The physical adsorption and covalent binding of ACF I onto the mica show very different surface topographies. The former exhibits the characteristic strand like structure with much less reproducibility, the latter displays a elliptic granular structure with better reproducibility, which suggests that the stability of ACF I molecules on the mica is enhanced by covalent bonding in the presence of glutaraldehyde. A small scale AFM amplitude mode image clearly shows that the covalently bonded ACF I molecule by glutaraldehyde has olive shape structure with an average size of 7 4 nm×3 6 nm×3 1 nm, which is very similar to the size determined from the crystal structure of ACF I. Anticoagulation factor I (ACF I) from the venom of Agki~strodon acutus is a binding protein to activated coagulation factor X (FXa) and possesses marked anticoagulant activity. Single ACF I molecule has been successfully imaged in air by tapping mode atomic force microscopy ( The AUR) with high resolution using glutaraldehyde as a coupling agent. The physical exhibits and covalent binding of ACF I onto the mica show very different surface topography. The former exhibits the characteristic strand like structure with much less reproducibility, the latter displays a elliptic granular structure. A small scale AFM amplitude mode image clearly shows that the covalently bonded ACF I molecule by glutaraldehyde has olive shape structure with a better reproducibility, which suggests that the stability of ACF I molecules on the mica is enhanced by covalent bonding in the presence of glutaraldehyde. An average size of 7 4 nm×3 6 nm×3 1 nm, which is very similar to the size determined From the crystal structure of ACF I.