In this paper, a new method called “projection kinematics” is presented for the determination of 3D configurations of DNA Origami Mechanisms (DOM) based on a single 2D projected image. The method enables finding all possible projected configurations of a DOM in space by solving the projection kinematics equations based on the minimum needed information measured from a 2D image. If redundant measurements are available, they can be exploited to eliminate ambiguous solutions. First, projection kinematic analyses were derived for the basic kinematic joints – revolute, prismatic, cylindrical and spherical joints and one degree-of-freedom (DOF) planar four-bar and two DOFs five-bar linkages. Then a generalized procedure for projection kinematic analysis is presented. Finally, a universal joint and a Bennett linkage fabricated by DNA origami self-assembly are chosen as proof-of-concept examples to verify the feasibility of projection kinematic analysis of DOM. This approach not only provides valuable guidance for the design of DOM, but also offers a low cost analysis approach for configuration estimation of spatial mechanisms when images from multiple viewing angles are not accessible, which is generally the case for the most commonly used analysis methods.