Atomic force microscopy (AFM), which provides true 3D surface topography, can also be used to determine the geometric parameters of proteins quantitatively at a single molecular level. In this paper, two different kinds of Escherichia coil MutS (MutS) protein were observed using AFM, and the geometric parameters of the proteins such as height, perimeter, area, and volume were measured. On the basis of these measurements, the molecular weight, association constant, oligomeric state, and orientation of MutS proteins on a mica surface were deduced. The oligomerization mechanism of MutS was analyzed in detail, and the results show that two different kinds of interactions between MutS protein may be involved in oligomerization. Our results also show that AFM imaging is an accurate method for analyzing the geometric structures of a single protein quantitatively at a single-molecule level.