[Background] Microbial protease has become the main source of industrial enzyme preparation. Screening microorganisms with special environmental adaptability has become a research hotspot. [Objective] To isolate and screen the strain with high protease activity from the alpine meadow soil on the Tibetan Plateau, and optimize the enzyme production conditions and explore enzymatic properties. [Methods] Strains were identified by morphology and molecular biological identification. The fermentation condition and enzymatic properties were studied by single factor and/or orthogonal test. [Results] Strain XC2 with high protease activity was identified as Bacillus subtilis. The optimum fermentation conditions were: soluble starch 4.0%, beef extract 1.0%, K+ 0.6%, 34 °C, pH 7.0, inoculum amount 2.0%, rotation speed 200 r/min after 13 h. The highest protease activity of XC2 reached 638.5 U/mL. The optimum reaction temperature and pH of the protease was 60 °C and 9.0 respectively. The enzyme activity is relative stable between 40 and 50 °C or pH 8.0?10.0. The protease activity of XC2 was significantly enhanced by Mn2+, but significantly reduced by Zn2+, Cu2+, Fe2+ and Fe3+. [Conclusion] Bacillus subtilis XC2 can efficiently produce alkaline protease, with good application prospect.