The development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a revolutionary step for genome engineering because it enables modification of target genomes. However, many biological applications with the CRISPR/Cas9 system are impeded by off-target effects and loci-dependent nuclease activity with various sgRNAs. Commonly used label-strategy-based CRISPR/Cas9 assays often suffer from possible disturbances to Cas9 activity and a time-consuming labeling procedure. Herein, we for the first time propose a DNA-templated CuNPs-based label-free CRISPR/Cas9 assay, with a low LOD of 0.13 nM and rapid detection in 35 min after CRISPR/Cas9 cleavage. Additionally, the site specificity of the DNA substrate was demonstrated. Through the proposed label-free strategy, a single-base change at a specific loci could lead to a significant reduction of the Cas9 cleavage effect, while the other common genetic modifications might be accepted by the CRIPR/Cas9 system. Therefore, the proposed label-free Cas9 assay may provide a new paradigm for the a priori in vitro CRISPR/Cas9 assay and exploration for in vivo biological applications.