Abstract: In order to expand the high-quality agarase categories, make full use of seaweed resources, and realize the efficient preparation of functional agar oligosaccharides, we derived a β-agarase from the marine bacterium (Agarivorans gilvus WH0801)(β-AGAase) by using genome mining technology, and predicted its physical and chemical properties as well as structural characteristics by bioinformatics analysis. Based on the results, β-AGAase is a non-secreted protein. The β-AGAase was extracellular expressed in Escherichia coli through the introduction of signal peptides by molecular biology methods, and its production efficiency was significantly enhanced through fermentation regulation strategy optimization. The optimal fermentation conditions are as followed: seed culture medium with an inoculation time of 5 h; fermentation starting medium was TB (pH 7.0); carbon source was 6 g·L⁻¹ of fructose; nitrogen source was 30 g·L⁻¹ of yeast extract II. After being cultured at 25 ℃ for 2 h, IPTG was added with a final concentration of 0.025 mmol·L⁻¹ for 48 h induction. Under these conditions, the obtained enzyme activity was 16.72 U·mL⁻¹ and was about 5 times higher than the initial enzyme activity, which proves that β-AGAase is of great potential for industrial application.