A gene, encoding a new alginate lyase AlgNJU-03, was cloned from marine bacteria Vibrio sp. NJU-03. The recombinant alginate lyase was characterized followed by being purified by NTA-Ni Sepharose. It exhibited the highest activity (6468.99U/mg) at pH 7.0 and 30°C. Interestingly, AlgNJU-03 possessed broader substrate specificity and high activity toward both polyM (poly β-d-mannuronate) and polyG (poly α-l-guluronate), indicating that it is a bifunctional alginate lyase. Furthermore, Km of AlgNJU-03toward polyG (4.00mM) is lower than those toward alginate (8.50mM) and polyM (10.94mM), demonstrating that the enzyme has a higher affinity to polyG. Meanwhile, the catalytic efficiency (Kcat/Km) toward polyG (11.47s−1/mM) is much higher than those toward sodium alginate (3.60s−1/mM) and polyM (0.50s−1/mM). ESI–MS analysis suggested that AlgNJU-03 mainly released disaccharides, trisaccharides and tetrasaccharides from the three kinds of substrates in an endolytic manner. Therefore, it may be a potential tool to produce alginate oligosaccharides with low DP.