We demonstrate a novel strategy of self-assembly to scale up origami structures in two-dimensional (2D) space using multiple origami structures, named “2D DNA jigsaw pieces”, with a specially designed shape. For execution of 2D self-assembly along the helical axis (horizontal direction), sequence-programmed tenon and mortise were introduced to promote selective connections via π-stacking interaction, sequence-complementarity, and shape-complementarity. For 2D self-assembly along the helical side (vertical direction), the jigsaw shape-complementarity in the top and bottom edges and the sequence-complementarity of single-stranded overhangs were used. We designed and prepared nine different jigsaw pieces and tried to obtain a 3 × 3 assembly. The proof of concept was obtained by performing the assembly in four different ways. Among them, the stepwise self-assembly from the three vertical trimer assemblies gave the target 2D assembly with ∼35% yield. Finally, the surfaces of jigsaw pieces were decorated with hairpin DNAs to display the letters of the alphabet, and the self-assembled 2D structure displayed the word “DNA JIG SAW” in nanoscale. The method can be expanded to create self-assembled modules carrying various functional molecules for practical applications.