The potential of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) copolymer production has remained unexplored for Cupriavidus sp. USMAA1020. This study is devoted to a full range exploration on the biosynthesis of polyhydroxyalkanoate (PHA) containing 3HV monomer, from the screening of carbon sources to the optimization of fermentation parameters. Fatty acid (oleic acid) and alcohol (1-pentanol) was found to be the best combination which resulted in high cell dry weight (CDW) (9.4 g/L), as well as high PHA accumulation of 76 wt%. Alcohols were found to be less toxic compared to other 3HV precursors; meanwhile, oleic acid contributed to sufficient amount of acetyl-CoAs as the building block for PHA thus contributing to high PHA content. Improvement was strategized by focusing on 1-pentanol concentration, incubation period and temperature which are the major factors affecting the copolymer production. An optimization study has resulted in high CDW (13.3 g/L) and PHA concentration (10.23 g/L), which increased significantly to 45 and 43%, respectively, compared to pre-optimized culture. This study warrants the usage of oleic acid and 1-pentanol as one of the viable strategies for industrial scale production of P(3HB-co-3HV) copolymer.