[Objective] The purpose of this study was to isolate a butanol-producing strain from the soil of corn field in Shiquan County, Shaanxi Province, China, and to improve its butanol tolerance and butanol yield. [Methods] Using the multi-factor complex screening and the treatment of butanol stress acclimation, the strain with a high yield and butanol tolerance was obtained. [Results] The results showed that through several batches of acclimation and screening, the mutant T64 was derived from isolated wild-type strain D64, its butanol tolerance was significantly improved and could grow well in the complex screening medium containing 20 g butanol/L. It produced 21.8 g/L total solvent (acetone, butanol, ethanol) and a butanol yield reached 15.18 g/L using 7% corn mash as fermentation medium, which was higher than that of the wild-type strain D64 (13.35 g/L). [Conclusion] In conclusion, the designed multi-factor complex screening is more effective than the mono-factor screening for obtaining high-producing butanol strain. In addition, the stress acclimation of increasing butanol concentration in long term provides new ideas to research the tolerance of butanol.