Crystalline nanostructures possess defects/vacancies that affect their physical and chemical properties. In this regard, the electronic structure of materials can be effectively regulated through defect engineering; therefore, the correlation between defects/vacancies and the properties of a material has attracted extensive attention. Here, we report the synthesis of Bi₂S₃ microspheres by nanorod assemblies with exposed {211} facets, and the investigation of the types and concentrations of defects/vacancies by means of positron annihilation spectrometry. Our studies revealed that an increase in the calcined temperature, from 350 to 400 ℃, led the predominant defect/vacancy densities to change from isolated bismuth vacancies (V_Bi) to septuple Bi³⁺-sulfur vacancy associates (V_BiBiBiSSSS). Furthermore, the concentration of septuple Bi³⁺-sulfur vacancy associates increased as the calcined temperature was increased from 400 to 450 ℃. The characterized transient photocurrent spectrum demonstrates that the photocurrent values closely correlate with the types and concentrations of the predominant defects/vacancies. Our theoretical computation, through first principles, showed that V_BiBiBiSSSS strongly absorbs ${\text{I}}_2(\text{sol})$, easily desorbs ${\text{I}}^{-}(\text{sol})$, and enhances the electrocatalytic activity of the nanostructures.