In-line digital holography is presented for the particles analysis in 3-D. Since the effects of zero order waves, conjugate image and defocused particle images degrade the reconstructed image quality of the focused particles during the reconstruction of in-line digital holography containing small particles at different depths, a numerical method for reducing these effects was proposed. A new subtraction method of reconstructed wave fields was presented for particles analysis. The effects of zero order term, conjugate image and the defocused particle images were simultaneously minimized. Therefore, the contrast of reconstructed amplitude images was improved. This method is useful to slice in-line digital holograms to display only in-focus particles at any particular plane. Furthermore, this process is accomplished from a single hologram without additionally required pre or post processing. The theory, simulation results and experimental examples were also given.