Enzymes are the biological macromolecules that speed up the conversion of substrates into products by lower the activation energy of the reacting species. Protein hydrolyzing enzymes (proteases) consist of the major assembly of enzymes being used in industry with a diversity of applications in the food, detergents, leather and meat industries. Due to increasing industrial demands, interest has been growing in microbial proteases for application in different commercial scale processes. Thus, there is need to explore new sources of alkaline proteases. The aim of this research was to identify potential protease producers. Out of six microbial strains Bacillus licheniformis and Aspergillus flavus were found to be the best producers of protease. To enhance the production of proteases by selected strains, different physical parameters, e.g., pH, temperature, inoculum size and fermentation time, were optimized by applying response surface methodology (RSM) under central composite design (CCD). Similary, nutritional parameters such as carbon and nitrogen source were also optimized and the influence of C:N ratio and metal ions on the yield of proteases, were also determined. The proteases obtained were purified by ammonium sulphate precipitation followed by dialysis, ion exchange and gel filtration chromatography methods. The purified proteases were immobilized by synthesizing cross-linked enzyme aggregates (CLEAs) and by optimizing the different parameters for CLEAs formation by RSM under CCD. SEM (scanning electron microscopy) of CLEAs were performed for the presence of protease in CLEAs. Free protease and CLEAs were compared by determining the effect of substrate (casein) concentration, pH and temperature. Free and CLEAs of proteases were used for industrial applications. Concerning industrial applications, alkaline proteases efficiently remove blood stain from fabric, remove the gelatin layer from used X-ray film, degrade animal bones protein, clot milk and dehair animal hides.