The pullulanase gene of thermophilic Bacillus subtilis WY-34 was cloned in E. coli to characterize the recombinant pullulanase. The pluA gene encoding pullulanase from the thermophilic B. subtilis WY-34 was overexpressed in E. coli. The properties of recombinant pullulanase were studied. Also substrate specificity of the recombinant pullulanase was evaluated in this paper. The pluA gene was 2.2 kb in length and encoded 718 amino acid protein. The recombinant pullulanase was purified to homogeneity using Ni-IDA agarose chromatography, resulting in a specific activity of 93.2 U/mg. SDS-PAGE and gel permeation chromatography analysis showed that the molecular weight of the protein is approximately 76.2 kD and 74.3 kD respectively. The purified enzyme was optimally active at 40 °C and pH 6.0, stable at 45 °C and retained more than 80% relative activity with in pH 6.0?9.0. It exhibited high substrate specificity toward pullulan. The properties of recombinant pullulanase may be useful for application of pullulanase in industry.