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Bacillus species are good secretors of proteins and metabolites. Many bacilli produce antibiotics and biologically active compounds, they are therefore used as probiotics in aquaculture. Most species of Bacillus strains have a high capacity to secrete a variety of extracellular enzymes such as amylase, arabinase, and cellulose as carbohydrates, lipase, several kinds of protease, xylanase, and also include many different penicillinases, nucleases, phosphatases, phospholipase C, thiaminase, and bacteriolytic enzymes. Alkaline Phosphatase is the most active of the enzymes, but also the most difficult to destroy at the end of the dephosphorylation reaction. In bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell membrane. Since this space is much more subject to environmental variation than the actual interior of the cell, bacterial alkaline phosphatase is comparatively resistant to inactivation,denaturation, and degradation, and also has a higher rate of activity.The present study demonstrate the applications of statistical design for media oprimization for production of ALP. The analysis revealed that peptone, glucose, and K₂HPO₄ significantly influenced alkaline phosphatase (ALP) production, with a 99.8% confidence level, identifying them as the most significant carbon, nitrogen, and phosphate sources, respectively.