Vibrio alginolyticus is a halophilic organism usually found in marine environments. It has attracted attention as an opportunistic pathogen of aquatic animals and humans, but there are very few reports on polyhydroxyalkanoate (PHA) production using V. alginolyticus as the host. In this study, two V. alginolyticus strains, LHF01 and LHF02, isolated from water samples collected from salt fields were found to produce poly(3-hydroxybutyrate) (PHB) from a variety of sugars and organic acids. Glycerol was the best carbon source and yielded the highest PHB titer in both strains. Further optimization of the NaCl concentration and culture temperature improved the PHB titer from 1.87 to 5.08 g/L in V. alginolyticus LHF01. In addition, the use of propionate as a secondary carbon source resulted in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). V. alginolyticus LHF01 may be a promising host for PHA production using cheap waste glycerol from biodiesel refining.