The extreme ultraviolet (EUV) molybdenum/silicon (Mo/Si) multilayers were fabricated using the direct current magnetron sputtering method on different preparation substrate: (a) wiped method, (b) untreated and (c) ultrasonic cleaning method. To meet the requirement of comprehensive characterization of morphology of substrates for EUV multilayer, a suitable combination of different measuring techniques, such as atomic force microscopy (AFM), x-ray diffractometer (XRD) rocking curve and synchrotron radiation (SR) reflectance, were chosen. It is shown that the reflectance of x-ray multilayers is sensitive to the roughness and cleanness of the substrate from the SR measurement results, and the maximum reflectance of 68.6% at 13.5nm was obtained using the ultrasonic cleaning method. It is demonstrated on the analysis according to the experimental results that the combination of surface characterization techniques using the AFM, x-ray scattering technique and SR characterization can be used for investigating the detail topography information of the substrate.