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Simple Summary: Proteases are vital enzymes that break down proteins into smaller units, peptides, or amino acids, playing a role in several biological processes. Due to various industrial applications, including detergents, laundry, leather, and pharmaceutical industries, the production of thermostable proteases is of great concern. Thermophilic bacterium Streptomyces thermovulgaris produced the metalloprotease in a bacterial expression system. Overexpression was achieved using an inducer in 4 h, yielding a soluble protease. Protein was purified by precipitation and an affinity chromatography system and was stable. It retained 80% activity at a wide range of pH and temperature with a half-life of 4 h, which is highly efficient for its use in various industries. Proteases hydrolyze proteins and reduce them to smaller peptides or amino acids. Besides many biological processes, proteases play a crucial in different industrial applications. A 792 bp protease gene (nprB) from the thermophilic bacterium Streptomyces thermovulgaris was cloned and expressed in E. coli BL21 using pET 50b (+). Optimal recombinant protease expression was observed at 1 mM IPTG, 37 °C for 4 h. The resulting protease was observed in soluble form. The molecular mass estimated by SDS-PAGE and Western blot analysis of the protease (NprB) fused with His and Nus tag is ~70 KDa. The protease protein was purified by Ammonium sulfate precipitation and immobilized metal ion affinity chromatography. The optimum pH and temperature for protease activity using casein as substrate were 7.2 and 70 °C, respectively. The mature protease was active and retained 80% of its activity in a broad spectrum of pH 6–8 after 4 h of incubation. Also, the half-life of the protease at 70 °C was 4 h. EDTA (5 mM) completely inhibited the enzyme, proving the isolated protease was a metalloprotease. NprB activity was enhanced in the presence of Zn2+, Mn2+, Fe2+ and Ca2+, while Hg2+ and Ni2+ decreased its activity. Exposure to organic solvents did not affect the protease activity. The recombinant protease was stable in the presence of 10% organic solvents and surfactants. Further characterization showed that zinc-metalloprotease is promising for the detergent, laundry, leather, and pharmaceutical industries. [ABSTRACT FROM AUTHOR]