Abstract: Caproic acid is a value-added product that has many uses in the preservation and synthesis of bio-energy. It is obtained via reverse β-oxidation reaction using electron donors and acceptors through the process of carbon chain elongation. The short-chain fatty acids are converted to high-value medium-chain fatty acids (such as caproic acid with six carbon chains). To improve the production yield of caproic acid, it is essential to clarify the relationship between reductase and energy supply, as well as the appropriate range of influencing factors and their mechanism in the biosynthesis process. This review paper describes the mechanisms of carbon chain elongation with lactic acid and ethanol as electron donors. Excessive ethanol oxidation, methanogenesis, and the lactate–acrylate pathways were introduced as competitive pathways during electron donor oxidation, and the corresponding inhibition methods were also reviewed. The reductase supply relationship between electron donor oxidation and electron acceptor reduction during the reverse β oxidation was discussed. In addition, this study clarified the utilization of energy by anaerobic microorganisms during the biosynthesis of caproic acid and two types of ATP synthesis: substrate level phosphorylation and electron transport phosphorylation. Electron bifurcation in the reverse β oxidation (a phenomenon in which two electrons from the same molecule are separated and redox potential is converted into energy to drive thermodynamically adverse reactions) and the role of different electron bifurcations in the production of caproic acid were evaluated. The influence of pH on the production of caproic acid driven by different electron donors was analyzed from the perspectives of competitive pathways, the growth range of functional microorganisms, and product inhibition. Regulating the collaboration between different bacterial communities and exploiting product separation techniques may enhance the production of caproic acid, and this should be investigated in the future. The role of CO₂ and H₂ as headspace in reverse β oxidation was investigated from the perspectives of substrates, competitive pathways, and thermodynamics. Relevant studies of the CO₂ loading rate and H₂ partial pressure were also reviewed. The development and current status of bioelectrochemical enhancement in the synthesis of caproic acid were examined, with emphasis on the fixation of CO₂. Future research should focus on synthesizing caproic acid using lactic acid as an electron donor and organic wastewater as a substrate by bioelectrochemistry. This review summarized the advantages and disadvantages of the biosynthesis of caproic acid, providing theoretical guidance on how to produce it and improve its yield.