Converting the widely present sulfide into elemental sulfur (S0) holds significant environmental protection and resource recycling value. Existing biotechnologies for producing S0 rely on regulating the O2/S or N O3− /S molar ratios, which face challenges of unstable yields under varying conditions. The complexity of sulfide oxidation process, involving numerous enzymes and diverse sulfur forms and valence states, poses a significant difficulty to understanding and advancing research in bio-S0 production. To facilitate progress in this area, this review re-examines the sulfur oxidation pathways with a focus on the synthesis, storage, and utilization of S0, considering both metabolic pathways and catalytic mechanisms. Integrating insights into the enzymes involved, including their structural features, subcellular location, active sites, electron transfer processes, and inter-enzyme synergy, were provided to enhance the understanding of sulfur metabolism mechanisms at a molecular level. Furthermore, several underexplored yet crucial areas were highlighted, including the identification and characterization of sulfur transmembrane transport proteins, the discovery and enrichment of microorganisms with defects in the sulfur oxidation genome (GD-SOMs), and the potential of genetic engineering in constructing GD-SOMs. Addressing these gaps provides valuable guidance for potential S0 production biotechnological innovations.