The aim of this research was to characterize the γ-PGA produced by Bacillus subtilis BL53, comparing the influence of cultivation time on the rheological and thermal biopolymer characteristics in order to identify its potential applications. Bacterial cultivations were carried out in bioreactors under previously optimized conditions (medium E, 37 °C, 2 vvm, and 1000 rpm) and the produced γ-PGA was extracted from culture broth at 48 and 96 h of cultivation. The biopolymer was characterized by measurements of the weight-average molecular mass (Mw) using static light scattering, melting (Tm) and deterioration (Td) temperatures, and rheological properties of the biopolymer solutions. There was no significant difference in Mw for biopolymers obtained in different cultivation times ((1.47 ± 0.30) × 106 g mol−1 at 48 h; (1.49 ± 0.23) × 106 g mol−1 at 96 h). Solutions of biopolymers obtained at 48 and 96 h presented Newtonian behavior, whereas the viscosity was higher for the 96 h samples, indicating a better interaction solvent–biopolymer. The thermal analysis showed Tm of 134 °C and 128 °C, whereas Td varied from 130 to 190 °C and 130 to 215 °C for 48 and 96 h samples, respectively. Tm peak area and fusion enthalpy were higher for 96 h biopolymer, indicating a higher crystallinity compared to 48 h γ-PGA. These characteristics of B. subtilis BL53 γ-PGA support the possibility of its use as thickener and functional ingredient in food, antitumor agent, and biological adhesive in medicine.