In this paper, we present layer-by-layer stacking method to fabricate self-assembled structures of block copolymers (BCP) toward the out-of-plane direction. Layer-by-layer stacking is realized by transferring a BCP film on one substrate to another. Specifically, a water-soluble polymer film is coated on the former substrate, which is placed and fixed in contact with a target substrate. Consequently, the BCP film is released from the substrate and transferred to the target substrate when immersed in de-ionized water. In our experiment, PS-b-PMMA is used to form and transfer self-assembled structures, and polyvinyl alcohol is used as a water-soluble polymer. We prepared two kinds of target substrates; one has horizontal cylindrical structures by BCP self assembly, and the other has groove structures by EB lithography. In the case of BCP patterned substrate, BCP film with vertical cylindrical structures is transferred onto the line structures of BCP. In the case of EB lithography patterned substrate, BCP film with vertical cylindrical structures is transferred in a doubly suspended condition. Furthermore, vertical and horizontal cylindrical structures are also observed to align along the grooves.