Poly(3-hydroxybutyrate) is a biodegradable and biocompatible polymer with many potential biomedical applications including medical implants and drug delivery systems. In this study, poly(3-hydroxybutyrate) production by Bacillus subtilis MTCC 9763 was optimized for different physical and chemical parameters. A 90% poly(3-hydroxybutyrate) accumulation was achieved with 2% fructose and 0.5% ammonium sulfate as carbon and nitrogen sources. The effective accumulation occurred at pH 7. The poly(3-hydroxybutyrate) content of dry cell weight increased to 87% at 36 h of incubation. The synthesized poly(3-hydroxybutyrate) was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopies. The oil-in-water solvent evaporation technique was used to fabricate the poly(3-hydroxybutyrate) microspheres. The surface morphology appeared smooth in the range of 1–2 µm with specific surface area ranging 19 to 20 m2/g. The synthesized poly(3-hydroxybutyrate) microspheres were nontoxic and exhibited good cytocompatibility toward NIH 3T3 fibroblasts.