Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that can cause serious illness and even death. L. monocytogenes mainly affects people who are predisposed due to an underlying immune system condition, such as cancer or AIDS, as well as other susceptible people like the elderly, pregnant women, newborn babies, or foetuses. The condition has flu-like symptoms, but it can lead to serious complications such meningitis, septicaemia, abrupt abortion, or neonatal listeriosis .L. monocytogens remains of great public health concern due to its high mortality rate and lack of accurate detection and identification methods used in the laboratories. However, the VITEK® 2 analyzer system was previously used for biochemical identification of many bacterial strains. Also, the ability of PCR-based method to identify the high-risk L. monocytogenes is an important strategy that could help in diagnostic of such pathogen in the laboratories. Therefore, in this study, the VITEK® 2 analyzer system and a PCR-based method for rapid and sensitive detection of L. monocytogenes in twenty samples of stool collected randomly from diarrhea patients were used. Our results detected the presence of L. monocytogenes in twelve stool samples, which conformed by VITEK® 2 analyzer system and PCR based-method. In more details, the results of VITEK® 2 analyzer system showed that twelve samples gave 91% matching to L. monocytogenes. In addition, the PCR results of the twelve samples gave clear bands with 320 bp in size (Amplicon length). We concluded that the VITEK® 2 analyzer systems is very important method for biochemical identification of L. monocytogenes, and the PCR technique is very specific and useful molecular method in diagnosis of L. monocytogenes infections.