Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserves. Bacillus subtilis can synthesize and accumulate PHA as carbon source under limiting conditions of nutrients. PHAs are promising candidates for the development of environment-friendly, biodegradable plastics. The aim of the present work was to study the optimization of PHAs using different carbon sources in the media and by using agro-industrial bio-wastes as a replacement for the carbon source. The study also identifies the potential of the strain to produce maximum dry cell weight and PHA from the cheap sources, i.e. waste samples taken from different industries like distillery effluent, sugarcane molasses, milk whey, and paper industry effluent. The use of these low-cost raw materials has the potential to reduce PHA production costs, because the raw material cost contributes a significant part in traditional PHA production processes. On studying the effect of different carbon sources in the medium, it was found that sucrose-supplemented Luria-Bertani medium produced maximum PHA production of 1.58mg/ml in 72h constituting 58.59% w/w of the total cell dry mass at a pH value of 7.0. By substituting the carbon source in the medium with biowastes from different industries, the maximal production of PHA was 2.5mg/ml PHA from 10% sugarcane molasses at the neutral pH range. The PHA produced in all the other industrial wastes was not comparable with that of sugarcane molasses. Hence, it can be concluded that sugarcane molasses can be used as a cheap source of carbon and is economical for the production of PHA on a large-scale.