Background: One of the most significant etiologic factors in acute gastroenteritis in children is adenovirus types 40/41. Objective: The aim of this research was to diagnosis of adenovirus type 40/41 in children with diarrhoea in three Cities in Iraq; Babylon, AL-Najaf and Karbala, and evaluation of serum concentration of Interferon gamma (IFN-γ), Interleukin (IL) 6 and 10. Patients and methods: A total of 450 stool samples were collected from children with diarrhoea, age range from 1 to 36 month. Adenovirus 40/41 has been detected in stool using Polymerase Chain Reaction (PCR) method. Serum concentration of IFN-γ, IL-6 and 10 have been measurement using Enzyme-linked Immunosorbent Assay (ELISA) technique.Results: Out of 450 stool samples, there were 150 Children (33.3%) infected with adenovirus 40/41, the highest rate of infection was in the Karbala City with 73 infections (16.2%) followed by Najaf and Babylon were recorded 55 (12.2%) and 22 (4.8%) respectively. Cytokines profile revealed a significantly substantial rise in viral patients compared to controls. IFN-γ, IL-6 and IL-10 levels in the age range of 1 to 36 months were higher than those in the control group at 69.5, 61.3 and 112.3 pg/ml respectively. Conclusions: There were high infections with adenovirus type 40/41 in Karbala City. Interferon-γ, IL-6 and IL-10 play an important role in infection which can be used in immunotherapy.