Three-dimensional (3D) alignment with 10 nm order accuracy in 3D electron-beam (EB) lithography has been achieved by means of highly accurate rotation control and mark location using the transmission electron signal. Accurately aligned EB writing from various directions on micron order resists blocks on a small substrate provides great structural flexibility in the creation of 3D nanostructures. As a demonstration of the accuracy, a 3D hydrogen silsesquioxane nanostructure was made by 3D EB writing and two-step development using different developers. Moreover, a 3D poly(methyl methacrylate) nanostructure was made by repeated 3D EB writing and development to demonstrate 3D nanofabrication with great structural flexibility.