A new waffle-like DNA origami assembly (DNA waffle) with nine nanometer-scale wells in a 3 × 3 matrix pattern has been successfully constructed and used as a scaffold for selective nano-patterning of individual protein molecules. The folding pattern of the scaffold was specially designed so that the dimensions of each well could be independently tuned according to the dimensions of the guest nanoparticles. We demonstrated that two distinct proteins, streptavidin (SA) tetramer (d = 5 nm) and anti-fluorescein antibody (IgG) (inter-paratope distance ∼ 14.0 nm), could be selectively captured in size-variable wells of dimensions 6.8 × 12 × 2.0 nm for SA and 6.8 × 12 × 2.0 nm or 10.2 × 12 × 2.0 nm for IgG, respectively, through the attachment of two biotins or two fluoresceins at the two edges of each well. This allowed the formation of a heterogeneous protein nanoarray of individual molecules. The position of SA or IgG capture can be fully controlled by placement of biotins or fluoresceins in the nanoarray well. Moreover, a hetero-nanoarray consisting of two kinds of enzyme: horseradish peroxidase-labeled streptavidin (HRP-SA) and alkaline phosphatase-labeled anti-FITC antibody (AP-IgG) was successfully constructed through selective attachment of biotin or fluorescein in any desired wells. Successful enzyme-heteroarray formation was confirmed by enzymatic activity analyses after purification of mixtures of enzymes and DNA waffles.