The present study focused on the screening and optimization of biopolymer polyhydroxybutyrate (PHB) production by Bacillus spp. using cost-effective substrates. Among 602 local Bacillus isolates, Bacillus thuringiensis B417-5 produced the highest amount of PHB (2.278 g/L, 60.07% of dry cell weight, DCW). 1 H NMR and FTIR analyses of the extracted polymer revealed the characteristic peaks of PHB. The optimization results showed that the highest PHB accumulation (2.768 g/L, 72.08% of DCW) was achieved when culturing B. thuringiensis B417-5 in a nitrogen-deficient medium containing 1% total sugar from sugarcane juice and 0.5% yeast extract, with a pH of 7.0 and an incubation temperature of 37 °C for 48 h. B. thuringiensis B417-5 can thus be considered a good candidate for large-scale production of PHB. We are reporting for the first time that sugarcane juice is a promising carbon source for economical PHB production by B. thuringiensis.