Objective A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized. Results Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 A degrees C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 A degrees C. Conclusion It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.