To fabricate large area soft X-ray multilayer with uniform optical properties, the thickness uniformity of single layer in a period structure must be well controlled. The method of single layer thickness uniformity analysis and control in magnetron sputtering system is established, and variable speed rotation of sample holder to realize the uniformity of large area soft X-ray multilayer is explained. Variable rotation curve is acquired by theoretical analysis. It is used in the variable speed rotation of sample holder to realize the uniformity of large area soft X-ray multilayer. The low angle X-ray diffraction (LAXRD) results reveal that LAXRD curves at different points of samples prepared by optimized variable speed rotation paths have nearly the same diffraction angles at the same diffraction order. This phenomenon proves that the multilayer thicknesses are nearly the same value. The calculation results indicate that the thickness non-uniformity of Mo layer can be controlled in 1.1% and Si layer can be controlled in 1.6% on an area with 200 mm diameter.