To examine the effect of the torsional constraints imposed on DNA substrates on Cas9 cleavage, we prepared constrained DNA substrates using a DNA origami frame. By fixing the dsDNA at the connectors of the DNA frame, we created torsionally constrained or relaxed substrates. We quantified the cleavage of constrained and relaxed substrates by Cas9 with qPCR. Moreover, we observed the Cas9/sgRNA complex bound to the DNA substrates and characterized the dissociation of the complex with high-speed atomic force microscopy. The results revealed that the constrained nontarget strand reduced the cleavage efficiency of Cas9 drastically, whereas torsional constraints on the target strand had little effect on the cleavage. The present study suggests that highly ordered and constrained DNA structures could be obstacles for Cas9 and additionally provides insights in Cas9 dissociation at a single molecule level.