In the biodegradation of aromatic compound pollution, dioxygenase is a key enzyme of many bacterial pathways. A naphthalene dioxygenase gene was identified and cloned from the Antarctic psychrophilic bacteria Shewanella sp.NJ49; the gene sequence, structure, functions, and expression have analyzed. A full-length sequence (924 bp) of dioxygenase gene was obtained, and the amino acid sequence of dioxygenase gene from Shewanella sp.NJ49 was similar to the catechol 2,3-dioxygenase (C23O) from Pseudomonas mendocina. The homologic identity was 99%. The expression level of dioxygenase gene by qRT-PCR in different habitats showed that NJ49 had the highest expression level when temperature was 15°C, naphthalene concentration was 50 mg/L, and the gene was under UV-B radiation for 4 h.