Acute gastroenteritis (AGE) is a gastrointestinal infection that most usually affects young children. Recently, the Enterovirus (EV) was added to the list of pathogens responsible for acute gastroenteritis. No previous studies in Iraq on the propagation of EV infection. This study's objective was to use Sandwich Enzyme-linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR) techniques to determine the prevalence of EV in children with AGE. To accomplish this, 120 stool samples were taken from Mosul hospitals in the period from October 2021 to January 2022 and the tested patients were within the age group of less than five years for both sexes. The results showed that the EV was detected in 16(13.33%) by ELISA and 26(21.67%) by PCR out of 120 stool samples. The sensitivity of ELISA was 53.85%, while the specificity was 97.87% when compared with PCR results. The ELISA and PCR both had a kappa value of (0.814) and an accuracy rate of (88.33%). Conclusions: Compared to PCR, ELISA has the advantage of being faster, less expensive, easier to perform, and appropriate for analyzing a large number of samples. It was very close to the PCR results. Therefore, it is possible to conclude that epidemiological studies and routine diagnostic procedures can both use ELISA to detect EV. The PCR technique is regarded as the gold standard for EV detection due to its high sensitivity and specificity.