Over the few years, there is an increased interest in the microbial enzymes to overcome its inability to meet the current and future requirements of the World. In the search of a kind, sucrase has a vital role to play with its variety of applications, particularly in the food and biofuel productions. In addition to their vast and varied applications, newer microbes are to be screened for sucrase production with their desirable properties. There are two saccharolytic enzymes induced by sucrose in Bacillus species which is encoded by SacB and SacA in the an extracellular levansucrase and an intracellular, respectively. In this experimental analysis, Plackett Burman Design was used for screening of nutrients for sucrase production by Bacillus subtilis. Sucrase activity was optimized by Plackett Burman design in Production Medium and then purified by column chromatography. Using MINITAB 15 Software, Sucrose, Yeast Extract, and Ferrous Sulphate had major source influence on sucrase activity compared to other components. In column purification, maximum amount of enzymes was obtained from the concentration of 0.5M NaCl-eluted sample.