This communication measures the inter-helical angle of the 10-23 DNAzyme-substrate complex by atomic force microscopy (AFM). Herein, we have devised a strategy to assemble the DNAzyme-substrate complex into a periodic DNA 2D array, which allows reliable study of the conformation of the 10-23 DNAzyme by AFM imaging and fast Fourier transform (FFT). Specifically, the angle between the two flanking helical domains of the catalytic core has been determined via the repeating distance of the 2D array. We expect that the same strategy can generally be applicable for studying other nucleic acid structures.