Preparation of large size, few-layered (FL) 2D materials is one of the challenging problems in material science. In this study, an anisotropic etching method to prepare FL (5–7 layer) MoO3 with width of tens of microns and length exceeding 100 μm on SiO2 was developed. Anisotropic etching along [100] direction was found to be accelerated in high concentration KOH solution. The first few layer on SiO2 surface kept intact in the first few seconds. The transformation of MoO6 octahedron to MoO4 tetrahedron was suggested to be due to the weak bonding of Mo–O(2) along [100] direction, while the etching speed of the first few layer was slowed down by the interfacial interaction. It’s shown that the surface defects were strongly affected by the etching time, and thus the electrical properties could be controlled.