Fibrinolytic enzyme production from Scopulariopsis brevicaulis OS 3456 isolated from a local soil sample was studied. The enzyme was purified by ammonium sulfate precipitation and gel filtration chromatography using Sephadex G-100, increasing its specific activity to 370 U/mg with a yield of 1.5% and a purification fold of 3.4. The molecular weight of the purified enzyme was 61.5 kDa determined by SDS-PAGE analysis. The optimum temperature of the enzyme was 37^oC, and it was stable over a pH range of 5.0–9.0 with maximum stability at pH 7.0. The activity was increased in the presence of ß-mercaptoethanol, Mn2+, Ba2+, triton X-100, and xylene by 137.1, 51.6, 41.4, 37.5, and 23%, respectively. Furthermore, the enzyme activity was inhibited by Cd2+, Al3+, EDTA, PMSF, and acetone. The in vitro thrombolytic activity of the undiluted purified enzyme (370 U/mg) was found to be 100%. Meanwhile, in the cases of 185, 92.5, 46.25, 23.125, and 11.562 U/mg, the clot lysis percentage was 76.8, 67.4, 57.8, 39.5, and 28%, respectively. A carrageenan-induced tail thrombosis model was applied to test the in vivo thrombolytic activity of the enzyme. The result indicated no obvious thrombus in the tails of mice treated with the tested enzyme (370 U/mg). However, when the enzyme was diluted, its thrombolytic activity decreased gradually. All these results explore the promising thrombolytic activity of the extracted fibrinolytic enzyme. Hence, more purification steps and more experimental animal studies are required in the future for its use as a commercial drug.