An effective RT-PCR method was developed to detect exogenous gene xylB transcript levels in Zymomonas mobilis CP4. Total RNAs without genomic DNA contamination were purified from wild-type and gene engineering strains, and were quantified to the same concentration. Then, cDNAs synthesis and PCR analysis of these samples were conducted by reverse transcription PCR. The optimal number of cycles was determined by observing amplification profile of target gene xylB and internal control gene 16s rRNA, and relative expression levels of xylB in various samples were analyzed by RT-PCR. The results indicated that the xylB transcript was not be detected in CP4, however that could be found in recombinant strains, in which xylB transcription abundance was similar. The enzyme assay furthermore confirmed that effective expression of the target gene. The method provided a useful and rapid tool for detecting transcript levels of target genes from various samples of Z. mobilis.