Pineapple is one of the most important agro-industrial sugar-based fruits in Thailand. In this study, the waste stream from pineapple cannery processing was utilised and evaluated for potential use in the production of a main biopolymer group widely known as polyhydroxyalkanoates (PHAs) through aerobic batch fermentation. Firstly, pineapple cannery waste (PCW) collected from three processing sources, pineapple juice (PAJ), peel and core juice (PCJ), and pulp-washing water (PWW), was used as a carbon source. Secondly, it was characterised and pretreated. Then, batch fermentation was performed by using the optimal condition (200 rpm agitation rate, 37 °C, and fermentation time of 72 h) under two different nutrient conditions in each type of carbon source. The results revealed that PHAs were produced during 24–72 h of fermentation without any interference. The PHAs product obtained was characterised by their properties. Interestingly, GC-MS showed homopolymer of poly 3-hydroxybutyrate (P3HB) group characteristics, such as OH, CH, and C=O; meanwhile, H1 NMR analysis showed signals corresponding to CH3, CH2, and CH, respectively. Remarkably, utilising the PCW showed a high-potential cheap carbon source for the production of PHAs as well as for the treatment of wastewater from the fruit industry.