Real-time quantitative PCR is a commonly used method to analyse the gene expression profile, it is important to select an appropriate reference gene for normalization of experimental data when using this method. In our study, we used two statistical methods to evaluate the gene expression stabilities of five reference genes (ldh, recA, rpoB, gapdh and 16S rRNA) under the different growth phases of Lactobacillus helveticus H9. The results showed that the best reference gene was ldh which was the most stable gene would be used for normalization of real-time quantitative PCR experiments data.