Aims: The agarase from Thalassomonas agarivorans BCRC 17492 was cloned and overexpressed in Escherichia coli. The characterization of the novel agarase was performed. Methods and Results: The genomic library of T. agarivorans BCRC 17492 was constructed for screening agarase gene. The novel b-agarase, namely AgaB1, was successfully identified and shared only 57% identity to reported agarase from Alteromonas sp. To characterize the AgaB1 protein, the recombinant AgaB1 can be obtained by heterologous expression in E. coli. The agarase activity of AgaB1 was achieved at 30Á25 U per mg at 35°C. According to the analysis of optimal conditions, the highest activity of AgaB1 was attained at 40°C, pH 7Á4 and 200 mmol l À1 NaCl, and half-life of AgaB1 can be maintained for almost 1 h at 40°C. Further determination of substrate hydrolysis indicated that AgaB1 had possession of both endo-and exolytic activity, and neoagarobiose was the major hydrolysis product by TLC and high-performance liquid chromatograph/mass spectrometer (HPLC/MS) analysis. Conclusions:We have successfully cloned and overexpressed the novel b-agarase from T. agarivorans BCRC 17492 in E. coli. The high yield and detailed characterization of recombinant AgaB1 was provided.