With the discovery of the significant medicinal value of alginate oligosaccharides and bioethanol produced by microalgae, alginate lyase has been the focus of research in all fields. Five alginate lyase genes in cluster from Vibrio alginolyticus were cloned and expressed in Escherichia coli. SDS-PAGE and enzyme activity showed that four of the five genes have the activity to degrade alginate. Optimization of the induction conditions, protein purification and enzyme properties of rAlgV3 with the highest enzyme activity were studied. The results showed that the enzyme activity of recombinant enzyme rAlgV3 increased from 2.34×10⁴ U/L to 1.68×10⁵ U/L, which was 7.3 times higher than before. The optimal reaction temperature was 40 °C, and the enzyme was relatively stable between 4 °C and 20 °C. The enzyme had a higher activity between pH 6.5 and 9.0, with the optimum pH 8.0. It showed a wide range of pH that the alginate lyase can exist stably between pH 4.5 and 9.5. Appropriate concentrations of NaCl and Fe²⁺, Fe³⁺ ions promoted enzyme activity. SDS and Cu²⁺ ions inhibited the enzyme activity. The enzyme degraded Poly-M fragments and Poly-G fragments, with a wide range of substrate properties. The degraded product of sodium alginate of rAlgV3 analyzed by ESI-MS mainly was oligosaccharides with a polymerization degree of 2 to 3, which means that rAlgV3 was an endo-type alginate lyase. This enzyme has the potential in the development of third-generation bioethanol and the production of alginate oligosaccharides.