A thermostable extracellular alkaline protease was isolated and purified 7.1-fold from Galium aparine using a sequential four-step procedure comprising ammonium sulfate precipitation, ion-exchange chromatography, ultrafiltration, and gel filtration. The purified enzyme, with a monomeric molecular mass of approximately ∼30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), exhibited optimal catalytic activity at pH 8.0 and 50 °C. It retained more than 65 % of its activity after incubation at 70 °C for 20 min, indicating high thermal and pH stability. The kinetic analysis revealed a K of 0.5 mM and a V of 63.63 μmol min mg, suggesting strong affinity between the enzyme and its substrate. Thermal stability studies showed that the enzyme followed first-order inactivation kinetics, with a half-life of 693.13 min at 50 °C and an activation energy (E) of 51.16 kJ/mol, confirming its thermostable nature. The catalytic activity was enhanced in the presence of Tween 80, while it was inhibited by ethylene diamine tetra acetic acid (EDTA), phenyl methyl sulfonyl fluoride (PMSF), SDS, and Triton X-100, suggesting it is a serine protease with metalloprotease-like features. The enzyme also demonstrated compatibility with various industrially relevant surfactants and solvents. These results underscore the potential of G. aparine-derived alkaline protease as a highly stable and effective biocatalyst, particularly for applications requiring enhanced stability and efficiency under processing conditions.