A new punched DNA origami assembly with periodic nanometer-scale wells has been successfully designed and constructed. Through the attachment of two biotins at the two edges of each well, just one streptavidin (SA) tetramer (d = 5 nm) was size-selectively captured in each 6.8 x 12 x 2.0 nm well; this allowed formation of a 28 nm-period SA nanoarray of individual molecules. The position of SA capture can be fully controlled by placement of biotins in the nanoarray well. Moreover, construction of a 2D nanoarray of individual SA tetramers through selective positioning of SA tetramers in any desired wells in a complex of such punched origami motifs is also possible. The stability of the SA captured by this fixation strategy (DNA wells and two biotin linkers) was directly compared on the same molecule with the stability of SA captured with other possible strategies that do not employ wells or two linkers. In this way, the robustness of this means of fixation was clearly established.