Photobacterium sp. TLY01, a novel strain of Photobacterium genus isolated from an oil field, was found to allow the accumulation of polyhydroxyalkanoates (PHAs) from a series of sustainable substrates, including glycerol, carbohydrates, plant oils, and volatile fatty acids. Interestingly, Photobacterium sp. TLY01 could effectively utilize plant oils or corn starch to synthesize poly-3-hydroxybutyrate (PHB). When propionate or valerate was added as secondary carbon source, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was produced. In fed-batch fermentation, the strain produced 53.89 g/L PHB using glycerol. When soybean oil and valerate were used as joint substrates, PHBV titer reached 16.28 g/L in bioreactor cultures. Furtherly, tryptone and yeast extract in culture medium were replaced with corn steep liquor to support PHA production. At last, a mixed substrate containing waste cooking oil, corn starch, and valerate was employed for PHBV production, achieving a titer of 4.01 g/L. The results indicated that Photobacterium sp. TLY01 is a promising strain for PHA production with low-cost sustainable substrates.