A series of Mo/Si multilayers with the same periodic length and different periodic number were prepared by magnetron sputtering, whose top layers were respectively Mo layer and Si layer. Periodic length and interface roughness of Mo/Si multilayers were determined by small angle X-ray diffraction (SAXRD). Surface roughness change curve of Mo/Si multilayer with increasing layer number was studied by atomic force microscope (AFM). Soft X-ray reflectivity of Mo/Si multilayers was measured in National Synchrotron Radiation Laboratory (NSRL). Theoretical and experimental results show that the soft X-ray reflectivity of Mo/Si multilayer is mainly determined by periodic number and interface roughness, surface roughness has little effect on reflectivity.