Abstract: Currently, little is known about a protocol in establishing the quantitative Real-Time PCR (qRT-PCR) system in gram-positive bacteria. Also, it is obscure whether the expression level of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (Cas9) in Listeria innocua is influenced by the pH. This study focused on providing a protocol for analyzing Cas9 gene expression in L. innocua at two pH conditions. The 37°C overnight culture was diluted 1:500 and grown in fresh Todd Hewitt Yeast extraction broth (THY) for 3 h. The collected pellet was washed two times in pH = 7.5 medium and was cultured for 4 h by using pH = 7.5 and pH = 6.0 medium, respectively. The culture was treated with RNA protection reagent for 5 min and the obtained pellet was suspended with mutanolysin for 30 min. Total RNA was extracted by using a kit and was incubated with DNase I, which was removed by adding phenol-chloroform-isoamyl alcohol thereafter. The purified RNA was precipitated via glycogen-ethanol treatment and was synthesized into cDNA. The 2‾ΔΔCt method was used to analyze the expression level of Cas9 gene with 16S rRNA gene as a reference. The gel electrophoresis showed that the quantity of 23S rRNA was about two-fold of 16S rRNA. The relative Cas9 expression levels of L. innocua in neutral and acidic medium were 1.08±0.49 and 1.66±0.54, respectively (p>0.05). Totally, the qRT-PCR system for analyzing Cas9 gene expression in L. innocua was successfully developed. The Cas9 expression level of L. innocua in acidic medium was similar to that in neutral medium.