The goal of this research was to isolate and identify the thermostable alkaline protease producing bacteria among several native Iranian microorganisms. At the end of screening program, a Bacillus subtilis BP-36 strain producing thermophilic alkaline protease was isolated from a hot spring in Ardebil province. The target enzyme was purified using a one-step Aqueous two-phase systems (ATPS) protocol involving 22% (w/w) polyethylene glycol (PEG)-10,000, and 18% (w/w) citrate with a yield of 39.7%, specific activity of 2600 U/mg and purification factor of 4.8. It was shown to have a molecular weight of 40 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified thermophile enzyme was stable in alkaline pH range (9.0-11.0) with the optimum pH of 9.0. It was highly stable at 60 °C and retained 100% activity even after 90 minutes, suggesting that it belong to the family of thermophilous. Collectively, our obtained data revealed that the thermophilic protease produced by B. subtilis BP-36 has the potential application in industrial processes under high temperature.