A psychrophilic bacterium Shewanella sp. NJ49 isolated from Antarctic sea-ice is capable of utilizing diesel fuel as the sole carbon and energy source. The gene encoding alkane monooxygenase (alkB) was obtained by using a genome walking technique in this strain, and revealed an open reading frame (ORF) of 1176 bp encoding a protein of 392 amino acid residues. Amino acid sequence alignment showed that its genetic relationship is close (99% identity) with Rhodococcus erythropolis PR4 (accession no. YP 002764193.1), Rhodococcus erythropolis SK121 (accession no. ZP 04388098.1) and Rhodococcus sp. Q15 (accession no. AAK97448.1). The alkB gene expression variations under different temperatures, various concentrations of n-Hexadecane, different handling time of UV-B radiation and high salinity were quantitatively analyzed by fluorescent quantitative real-time PCR (qRT-PCR). Finally the optimized expression levels were obtained, respectively. These results indicate that alkB gene as a functional gene likely play a crucial role in Shewanella sp. NJ49 degrading petroleum hydrocarbon under Antarctic extreme environments.