Abstract: Objective To provide the effective strategy for genome annotation and transcription regulation by studying the strandspecif ic deep sequencing of transcriptome in Pseudomonas aeruginosa. Methods RNA was extracted from Pseudomonas aeruginosa PAO1.After rRNA was removed, the RNA was inversely transcribed into cDNA with specific primer and sequenced by PCR amplif ication.The data were analyzed using Bowtie and rSeq software. Results Of the 26, 557, 654 trimmed read fragments (except for 209, 928 no-valid read fragments), 26, 347, 726 were the genome matching read fragments, indicating that the sequencing could cover almost 99% of the genomes.Seventy-eight point seven percent of the genome were the mapped reads on both strands.The most highly represented gene was ssrA and 2283 new annotation genes were discovered. Conclusion Strand-specif ic deep sequencing of transcriptome is successful in Pseudomonas aeruginosa.