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This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from <i>Bacillus stearothermophilus</i> isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 10⁸ CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of <i>Bacillus</i> strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca²⁺/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca²⁺ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.