Protein catalysts called enzymes speed up the pace at which chemical processes approach equilibrium. This study presents a comprehensive comparative analysis of enzymes extracted from (sterculia foetida) java olive, a plant species known for its diverse bioactive compounds. The isolation of this enzyme was done from a fruit seed (S. foetida). After the fruit seed was crushed, and the extract was extracted using mortar and pestle, an enzyme assays were used to determine the presence of protease enzymes. The enzymes of interest were precipitated using ammonium sulphate solutions (60%). After crude extract has been produced and followed by the partial purification by dialysis method. The partially purified seed samples were subjected to Lowry’s technique for protein estimation and enzyme activity. To find the enzyme peak activity, a series of biochemical characterization, the maximum activity of protease at optimum pH and temperature were determined respectively. For various substrate concentrations, including gelatin, casein and skimmed milk, the apparent Vmax and Km values were calculated. Furthermore, (Native PAGE) was employed for the separation and visualization of proteins within the enzyme extracts. The kinetic properties of protease were determined by Michealis – Menten Kinetics, Lineweaver burk Kinetics was studied.