In this study, the effect of important parameters such as the pixel size and number of a CCD, the object distance, the wavelength of laser, and the particle diameter on the depth of focus in digital in-line particle holography were investigated. The depth of focus in several different cases was calculated using simulation holograms and detailed description of the depth of focus in digital particle holography was presented. The depth of focus is directly proportional to the object distance and the particle size. With the increase of the wavelength of laser, the depth of focus is decreased. The depth of focus is also inversely proportional to the pixel size and number of a CCD. Using the data of depth of focus from simulation holograms and a data-fitting software, we obtained the prediction equations of depth of focus for typical CCD cameras. Finally, the prediction equations of depth of focus in digital particle holography were verified by investigating real holograms of the calibration target in different cases and satisfied agreement between measured values and predicted values was confirmed.