This study investigated the purification and biochemical characteristics of the protease produced by Pediococcus pentosaceus isolated from Harbin dry sausages. The optimized fermented conditions were as follows: fermentation time 36 h, initial pH 5 and fermentation temperature 30 °C. A 29.6 kDa extracellular protease was purified using ammonium sulphate deposition, ion exchange layer system and gel filtration. The protease produced by P. pentosaceus had a certain pH and thermal stability at pH 6 and 30 °C. The microbial protease activity could be inhibited by ethylene diamine tetraacetic acid disodium salt (EDTA). Vmax and Km of the protease were 43.9 mg/min and 8.3 mg/mL, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) reflected the ability of the protease to hydrolyse sarcoplasmic and myofibrillar proteins, particularly those of myosin heavy chain, paramyosin, actin, phosphorylase and creatine kinase-M types. 3D structure modelling of the P. pentosaceus protease found two domains in the protease protein and the correlation of the active sites with protease properties and substrate specificity. In conclusion, P. pentosaceus can be used as a starter culture or enzyme producing strain for inoculation in Harbin dry sausages.