A technique based on off-axis digital holographic microscopy is proposed to measure three-dimensional position of particle in a liquid environment. The technique uses the transmitting off-axis digital holographic microscopy system, and the method combines the optical path length (OPL) difference method and fringe removing method. The OPL difference method is used to measure the axial position of the particle by locating the inflection point of OPL difference curves of two specific points in the particle. The fringe removing method is utilized for the off-axis holograms to measure the in-plane displacement of the particle with frequency filtering and Hough transform combined. Compared to the traditional digital holographic particle tracking techniques, the OPL difference method applies the OPL between two points to relative calculation instead of plane calculation. As a result, the measurement efficiency is improved, auto-focusing of particles is not needed, and the particle position is not constrained by the defocusing plane. The experimental results of nano-scale three-dimensional particle measurement show that the resolution ability reaches nano-scale when the OPL difference method is combined with the fringe removal method to measure the three-dimensional displacement of particles in the liquid environment.