[Objective] Considering of the extremely special biochemical characteristics of Acidithiobacillus sp., the efficient double-layers screening, fed-batch high-density fermentation strategy and preservation method was respectively established and optimized for enhancing the utilization and reservation efficiency of these microbial resources. [Methods] The heterotrophic microorganisms such as Sacchromyces ellipsoideu and Rhodotorula sp. was employed as bottomed culture when the transmission electronic technology (TEM) was employed for observing the morphological differences. Based on the designed sulfide medium and elemental sulfur fed-batch strategy, the logarithmic phase of Acidithiobacillus thiooxidans was extended when the specific growth rate was improved. The effects of different preserved methods on cell survival rates were also investigated. [Results] Using heterotrophic microorganisms——Rhodotorula sp. as the underlying culture in the double-layers culture, the screening cycle was reduced by 1/3, while the plating efficiencies of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were improved by about 3 times. More regular cell morphology was shown by TEM in the double-layers culture. The average specific growth rate of Acidithiobacillus thiooxidans was improved via fed-batch fermentation strategy in the Starkey-sulfide medium. The biomass conversion rate of sulfur and productivity were improved by 31.1% and 187.9% respectively, compared to the batch fermentation. The simple and efficient 4 °C cryopreservation perseveration was more suitable for preserving Acidithiobacillus sp. with the validity period about 1-3 months. [Conclusion] The screening efficiency of Acidithiobacillus sp. could be effectively improved by the double-layers culture with the auxiliary culture-Rhodotorula sp.. High-density fermentation of Acidithiobacillus thiooxidans was achieved via fed-batch fermentation strategy in the Starkey-sulfide medium. Simple and efficient 4 °C cryopreservation preservation was more suitable for preserving Acidithiobacillus sp. in the short-term.