High resolution three-dimensional (3D) position of microspheres tracking is the key point in the aero of single molecule force spectrum. Digital holography is emerging as a promising optical technique for 3D microspheres tracking. A sequence of holograms are acquired using charge couple device with an in-line digital holographic microscopy. Each hologram is subsequently reconstructed with Rayleigh-Sommerfeld propagation function and then deconvolved it with its propagator to suppress the artifacts and blur. Also polynomial fitting is introduced to fit the intensity profile of the volumetric reconstruction in the Z direction which can improve the resolution of 3D microspheres tracking. As a result, this method can measure multiple microspheres simultaneously and also can measure the overlapped spheres. The resolution in the Z direction is approach 2 nm, this is significant to the study of single biological molecule dynamics and particles image velocimetry etc.