[Objective] In view of the difficulty in the culture of chemoautotrophic bacteria, this study analyzed the reasons for the difficulty based on the theory of electron distribution and explored the feasibility of using the electron distribution strategy for increasing the biomass of chemoautotrophic bacteria based on pure culture. [Methods] From the perspective of maintaining intracellular pH balance and optimal energy metabolism, we calculated the optimal distribution ratios of electrons produced by the sulfur-oxidizing bacterial strain Halothiobacillus sp. DCM-3, nitrite-oxidizing bacterial strain Nitrobacter sp. N1, and ammonia-oxidizing bacterial strain Nitrosomonas sp. SCUT-1 to O₂ and CO₂ by oxidizing corresponding substrates. Furthermore, different molar ratios of O₂ to HCO₃^- (CO₂) were set respectively to form different electron distribution ratios for pure culture verification of the strains. Substrate and product concentrations were measured by ion chromatography and ultraviolet spectrophotometry, and cell density was measured by the dilution coating method. [Results] The optimal electron distribution ratios of strains DCM-3, N1, and SCUT-1 were 0.733:0.267, 0.867:0.133, and 0.6:0.4, respectively. Based on the optimal electron distribution ratios, strains DCM-3, N1, and SCUT-1 could synthesize 3.967 ATP/S₂O₃^2-, 0.433 ATP/NO₂^-, and 1.35 ATP/NH₃, respectively. According to the calculation results, the main reasons for the difficulty in culture were the small amount of ATP synthesized with the energy provided by per unit substrate and the need to control a low oxygen concentration and supplement an appropriate amount of inorganic carbon. The results of pure culture verification showed that the biomass of DCM-3 under the optimal ratio was 6.5×10⁷ CFU/mL, which was 2.2 times that of the control group. The biomass of N1 under the optimal ratio was 7×10⁶ CFU/mL, which was not significantly different from that of the control group. However, the HCO₃^- concentration (0.4 mmol/L) of the optimal ratio of strain N1 was significantly lower than that (2.5 mmol/L) of the control group, which meant that the strain showed a growth characteristic of tending to higher oxygen concentration but lower CO₂ demand, which was consistent with the calculated optimal ratio. The biomass accumulation per unit NH₄⁺ concentration of SCUT-1 strain in the group with controlled O₂ and CO₂ was more than 1.3×10⁶ CFU/(mL·(mmol/L)), which was 25%-40% higher than that obtained under sufficient O₂ and CO₂. [Conclusion] The culture strategy of chemoautotrophic bacteria based on electron distribution restricts the culture conditions of electron distribution by limiting the molar amounts and forming a certain ratio of O₂ and CO₂, which helps to improve the biomass accumulation under the same substrate condition and provides certain strategic reference for the culture of chemoautotrophic bacteria.