Particle tracking technology is one of the most important research tools for dynamic process in biology. To obtain the relative biology information, three-dimensional position measurement of micro-nano scale particles is needed. During the actual measurement, due to the interaction force between particles and the interaction force between particles and other in the liquid environment, the overlap phenomenon of the particle patterns is unavoidable. Overlapped particle patterns lead to the feature cross of rings, and the feature is hard to be recognized by traditional methods. So we can not obtain the position of the particles. In order to solve this problem, holographic reconstruction focusing method (HRFM) is proposed, as it can measure three dimensional position of particles by accurately locating their horizontal position. According to the experimental results, HRFM for locating the position of particles is more accurate than traditional method, especially for the measurement of overlapped particle position. The three-dimensional displacement resolution of the proposed method for single particle measurement and overlapped particles measurement can both reach 2 nm.