Objective The purpose of this study was to establish a rapid method for synthesis and detection of guild RNA (gRNA) which is an essential component in CRISPR/Cas9 knockout technology. Methods First, the Nkp46 gRNA core fragment was synthesized as amplification template. Second, the forward and reversed primers of the matched gRNA were designed using the Nkp46 gene as reference sequence. Third, the DNA fragment of Nkp46 gRNA was amplified by PCR technology using the synthesized gRNA core fragment as template. The gRNA was reversely transcribed in vitro using amplified DNA fragment as template. The efficiency and specificity of gRNA and its interaction with Cas9 were detected in vitro. Results The specificity and activity of Nkp46 gRNA were high. The obtained gRNA interacted with Cas9 enzyme and successfully cut the target double-stranded DNA at the designed site. Conclusions The method for synthesis and detection of gRNA established in this study is faster than the original method, and the created gRNA is fully functional.