The aim of the study is to optimize the cultivation conditions of Bacillus subtilis to increase the activity of α-amylase in the culture fluid by selecting the concentration of calcium chloride (CaCl2), pH of the medium, and the duration of cultivation. The study was carried out in 2023–2024 using the B. subtilis strain from the Museum of Microbiological Cultures of the Department of Microbiological Synthesis Technologies. Cultivation was carried out by the submerged growth method in an L-broth medium at 37 °C for 48; 108 and 168 h. According to the design of a complete factorial experiment, the concentration of CaCl2 (1.0; 3.0; 5.0 g/l) and pH of the medium (4.0; 5.0; 6.0) were varied. α-amylase activity was determined spectrophotometrically using the iodine-starch method. Each experiment was performed in triplicate. A mathematical model was constructed as a first-order polynomial equation, adequately describing the dependence of enzyme activity on the studied factors. Analysis of the regression coefficients revealed the greatest positive effect of factor X2 (CaCl2 concentration, coefficient +2.75). The highest activity within the experimental matrix (13.08 U/ml) was observed at pH 6.0, CaCl2 concentration of 5 g/l, and duration of 168 hours. The negative value of the linear coefficient for pH (–1.68), combined with strong interaction effects, indicates a complex, nonlinear effect of medium acidity on enzyme synthesis. Based on model analysis and the steepest ascent method, optimal B. subtilis cultivation parameters for α-amylase synthesis were determined over the studied range: pH 6.0, CaCl2 concentration of 5 g/L, and duration of 168 h. Calcium chloride concentration had the greatest positive effect on enzyme yield. The model predicts that further optimization could significantly increase α-amylase activity beyond the studied range of factors.