CRISPR/Cas9 is a highly efficient genome engineering tool, yet its off-target effects and sequence-dependent cleavage activity across different sgRNAs remain major concerns for its application. Here, we propose a nicking triggered exponential amplification reaction (NTEXPAR), a fast and sensitive in vitro method, to detect the double strand DNA cleaved by down to 10 pM Cas9 with a linear range of 100 pM to 20 nM. With this newly developed amplification method, Cas9 cleavage activity can be quantified in 40 min and the optimal sgRNA design for specific target sequence can be successfully determined. Using the pre-screened sgRNA, we are able to distinguish single nucleotide mismatch in a gene silencing experiment. This fluorescence based isothermal assay provides a versatile tool for the pre-screening of sgRNAs to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.