Establishing a rapid, accurate, high-throughput and easily implementable genotyping method is highly desirable for functional genomics, genetic improvement and mutant screening. Here, we describe a convenient and inexpensive technique for genotyping using the targeted DNA endonuclease activity of Cas9 or Cas9NG ribonucleoproteins complex (sgRNA/Cas9-RNP or sgRNA/Cas9NG-RNP). In this study, Cas9 and Cas9NG protein purified from E. coli extract was assembled with in vitro transcribed single guide RNA (sgRNA)or enhanced sgRNA (esgRNA) as an assembled ribonucleoprotein (RNP) complex to fulfill the targeted endonuclease activity on the PCR amplicons of ZmWx exon 7. The restriction profiles can be converted into genotyping results of wildtype, homozygous or heterozygous mutant, respectively. Our data showed that ZmWx gene-edited mutants can be genotyped rapidly and efficiently by the sgRNA-optimized system esgRNA/Cas9. The reaction component optimization data suggested that 500 ng of DNA substrates could be cleavaged completely by incubating with 1 µg 1:1 molar ratio of esgRNA/Cas9 ribonucleoproteins for 30 minutes at 37°C, or by 4 µg 1:1 molar ratio of esgRNA/Cas9NG ribonucleoproteins for 4 hours at 37°C. Expanding the targeting flexibility of mutant detection via esgRNA/Cas9NG indicated that Cas9NG variant might recognize relax NG PAM (protospacer-adjacent-motif, PAM) at the expense of decreasing restriction activity, which is necessary to improve the activity of esgRNA/Cas9NG by further optimization. Therefore, the establishment and application of esgRNA/Cas9 based PCR/RNP technique provides an easy, simple and low-cost approach to genotyping in functional genomics, molecular breeding and mutant screening. In addition, our in vitro data on esgRNA/Cas9NG has certain and significant reference value for developing it into in vivo genome editing studies.