The resolution optimization and sampling strategies in practical digital in-line holography (DIH) with spherical wave illumination are analytically studied by optimizing system parameters. Optimal parameters of holographic recording, including illumination wavelength, numerical aperture of the spherical illumination wave, source-sensor distance, object–sensor distance, and sampling parameters of the sensor, for achievable resolution are worked out. A formula is derived to guide the DIH system design in general cases. Different sampling strategies associated with corresponding reconstruction waves (plane wave and spherical waves with various curvatures) are analyzed. The reason why recording with spherical wave and reconstructing with plane wave works well in practice is explained in detail. We also present how to determine the reconstruction distance and magnification to reconcile the curvature difference. The analysis is carried out mathematically and verified by simulated holograms. Optical experiments with U.S. Air Force resolution target are carried out based on the analysis for further verification.