Polyhydroxyalkanoates (PHAs) are three-level group of biodegradable polymers and attractive substitutes over conventional plastics to avoid the pollution problems. The yeast strain isolated from sugarcane juice, identified as Wickerhamomyces anomalus VIT-NN01, was used for the production of polyhydroxyalkanoates (PHA). Response surface methodology (RSM), three-level six variables Box-Behnken design (BBD), was employed to optimize the factors such as pH 8.0, temperature 37° C, sugarcane molasses (35 g/L) supplemented with co-substrate palm oil (0.5%),corn steep liquor (2%) after a period of 96 h of incubation for the maximum yield (19.50 ± 0.3 g/L) of PHA. It was well in close agreement with the predicted value obtained by RSM model yield (19.55 ± 0.1 g/L).Characterization of the extracted polymer was done using FTIR, GC–MS, XRD, TGA and AFM analysis. NMR spectroscopic analysis revealed that the biopolymer was poly (3-hydroxybutyrate-co-3-hydroxyvalerate), copolymer of PHA. This is the first report on optimization of PHA production using yeast strain isolated from natural sources.