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This study aimed to screen alpha-amylase producing microorganisms from honey as a low water activity medium, a suitable source for selecting stable and cost-beneficial bacterial enzyme production systems. Plackett-Burman method was used to select twelve effective factors including pH, inoculum size, temperature, time, corn starch, KH 2 PO 4 , peptone, MgSO 4 , CaCl 2 , NaCl, glycerin, and yeast extract concentrations on bacterial alpha-amylases production yield. The Box-Behnken method was utilized to optimize the level of selected significant factors. The stability of bacterial alpha-amylases was also determined in low pH and high-temperature conditions. In addition, in silico study was used to create the alpha-amylase structure and study the stability in high-temperature and low water available condition. Among all isolated and characterized microorganisms, Bacillus megaterium produced the highest amount of alpha-amylases. The in silico data showed the enzyme 3D structure similarity to alpha-amylase from Halothermothrix orenii and highly negative charge amino acids on its surface caused the enzyme activity and stability in low water conditions. Based on Box-Behnken results, the temperature 35 °C, pH 6 and starch 40 g/l were determined as the optimum level of significant factors to achieve the highest alpha-amylases unit (101.44 U/ml). This bacterial alpha-amylases enzyme showed stability at pH 5 and a range of temperatures from 40 to 60 °C that indicates this enzyme may possess the potential for using in industrial processes.