Listeriosis is the third most common food-borne illness and is considered one of the most dangerous bacterial zoonotic infections globally. This research aimed to isolate and characterize Listeria monocytogenes (L. monocytogenes) from diseased sheep in Egypt. A total of 240 samples collected aseptically from sheep were subjected to isolation and biochemical characterization of L. monocytogenes. L. monocytogenes are Gram-positive rods that are both aerobic and facultatively anaerobic. Thirty-one listeria isolates (12.9%) were recovered from diseased sheep. All tested isolates were positive for catalase, Voges-Proskauer, carbohydrate fermentation, esculin, gelatin hydrolysis, and methyl red tests. Meanwhile, they were negative for oxidase, indole, urease, and nitrate reduction tests and did not utilize citrate in the citrate utilization test. Listeria isolates produced yellow butt/yellow slant on the TSI agar medium with no H₂S or gas production. Therefore, this work focused on the isolation of L. monocytgenes from diseased sheep and biochemical characterization of the recovered isolates using traditional and Microbact™ 12L listeria identification systems.