To improve the resolution of three-dimensional position measurement of micrometer scale spheres in liquid, especially the resolution in the axial axis, a method named three-dimensional microsphere tracking using off-focus images based on cross-correlation matching algorithm is developed. It is a combination of the cross-correlation algorithm which has high precision matching characteristics to similar images and the idea of tracking three-dimensional microspheres using off-focus images. As a result, the experiments show that a standard deviation of about 0.64 nm is reflected when tracking the same microsphere continuously in the axial direction in four minutes, and a resolution of 1 nm step measurement in all three axes is achieved. This is significant to the study of single biological molecule dynamics and particle image velocimetry etc. Under the same experimental conditions, the comparative measurement and analysis of three-dimensional microsphere tracking with digital in-line holographic microscopy show that the method exhibits high feasibility and resolution in practical measurement applications.