<p>Lipase-catalyzed transesterification of waste oils offers a greener alternative to conventional chemical routes for biodiesel production. In this study, a lipase from <i>Pseudomonas aeruginosa</i> was cloned, heterologously expressed, partially purified, and immobilized in calcium–alginate beads for the transesterification of waste cooking oil. Following ammonium sulfate precipitation and dialysis, the enzyme achieved a maximum specific activity of 6,595.7 U·mg⁻ ¹ in the dialyzed fraction. SDS–PAGE analysis revealed a single prominent band at approximately 37 kDa. Immobilization in 2% (w/v) sodium alginate produced uniform, mechanically stable beads (~2 mm) that retained catalytic activity and functioned as heterogeneous biocatalysts. The enzyme exhibited an alkaline activity profile, with maximum activity at pH 8.0 and measurable activity across pH 7.0–8.5. Arrhenius analysis of temperature-dependent activity (20–60 °C) yielded an apparent activation energy of 51.3 kJ·mol⁻ ¹ (R² = 0.94), indicating moderate temperature dependence. Under optimized reaction conditions (oil: methanol molar ratio 1:3, 15 g immobilized enzyme per 100 mL oil, 37 °C, 48 h, 180–200 rpm), a fatty acid methyl ester (FAME) yield of 68.43% was obtained. These results demonstrate the feasibility of using an immobilized P. aeruginosa lipase for the enzymatic conversion of waste cooking oil to biodiesel.</p>