Fe/Ru multilayers were prepared by high vacuum e-gun evaporation system and the samples were studied by HRTEM and VSM. The interface structure of Fe/Ru multilayers with different thickness and its effects on magnetic properties were analyzed. Results show that the Fe/Ru multilayers have good periodic structure. When Fe layer is very thin, Fe layer is not continuous and the multilayer show a superparamagnetic property due to the Fe particles distributed as islands. HRTEM of Fe/Ru interface shows the difference in roughness between the two interfaces of Fe/Ru. The origin of the positive magnetoresistance (MR) is related to the difference in roughness, in which the scattering spin asymmetry is less than one. After annealing in vacuum, the negative MR of multilayer is increased greatly, indicating that it is originated from the roughness of the interlayer.