Aims: This study aims to detect the effect of nano-sized calcium carbonate as an ingredient in growth medium on the production of alkaline protease by Streptomyces spororaveus. The proportional relationship between highly production of alkaline protease and calcium carbonate nanoparticles emphasizes the unique and super properties of nanotechnology that applied in all field. Methodology and results: The high production of protease from S. spororaveus accompanied with presence of calcium carbonate nanoparticles as one of growth medium's constituents. Both qualitative and quantitative tests for proteolytic activity proved this fact. Agar-well diffusion method revealed that, the proteolytic activity with calcium carbonate nanoparticles (45 mm) is higher than that with calcium carbonate (30 mm). Calcium carbonate nanoparticles led to 150 μg/mL of protease, while calcium carbonate led to 65 μg/mL only. The crude protease was purified by ammonium sulphate precipitation and gel filtration column chromatography using Sephadex G-100. The purified protease was separated by SDS-PAGE as a single band at 30 kDa. The highest proteolytic activity was obtained at pH 8.5 and 45 °C as optimum environmental conditions. The purified protease has inhibited the growth of Candida albicans ATCC-10231 and Aspergillus brasiliensis ATCC-16404 at 8 mm and 10 mm of inhibition zone respectively. Conclusion, significance and impact of study Calcium carbonate nanoparticles in the composition of starch nitrate broth is good stimulus for highly proteolytic activity of S. spororaveus. Shake-flask fermentation method proved that, the concerned protease is an alkaline and thermostable up to 70 °C. However, the best pH and temperature values are 8.5 and 45 °C, respectively. This study can be applied to manufacture a modified starch nitrate broth medium for highly production of proteases from Streptomyces bacteria.