Abstract Background: Verotoxins are bacterial virulence factors produced by E. coli O157:H7, transmitted by the fecal-oral route. Objectives: The aim of this article was to diagnose E. coli O157:H7 which causes diarrhea and sometimes develops into HUS, which considers pig health problems and estimates the levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α in the sera of pediatric patients infected with Enterohemorrhagic E. coli compared to the control group. Materials and Methods: Stool and blood samples were collected from 421 pediatric patients with diarrhea, ranging in age from birth to 13 years old, from March to October 2022. Samples were collected from Al Noor Teaching Hospital, Babylon Hospital for Pediatric and Gynecology, Hilla, Iraq. E. coli O157:H7 was cultured on eosin methylene blue (EMB) and Sorbitol MacConkey agar (SMA), confirmed by biochemical test and cultured on HiCrome E. coli O157:H7 selective medium which was an agar base supplemented with cefixime tellurite agar. Serum from 30 pediatric diarrhea patients infected with E. coli O157:H7 compared with 30 healthy children as control group used to determine serum levels of IL-8 and TNF-α by sandwich ELISA. Results: The results revealed that out of the total 421 samples used in this study, E. coli O157H:7, represented 7% (30 of 421) stool samples. This 30serum samples of infected children as well as 30 samples from healthy children subjected to the estimate serum level of IL-8 and TNF-α which record significant differences P ≤ 0.01 and P ≤ 0.05 to this cytokines in different age group; the mean of IL-8 level was 283.62 ± 17.8 pg/mL (7–9 years), and the mean of TNF-α was 208.62 ± 28.7 pg/mL (10–13 years) comparative with the control group of 80.58 ± 15.4pg/mL and 32.50 ± 7.5 pg/mL, respectively, and also result showed an increased mean level of IL-8 than TNF-α in the male comparative with female 195.19 ± 10.4 pg/mL and 159.05 ± 12.4 pg/mL, respectively, comparative with the control group. The result showed no significant differences in IL-8 and TNF-α between watery diarrhea (192.43 ± 24.3 pg/mL and 136.05 ± 20.4 pg/mL) and bloody diarrhea (189.02 ± 22.5 pg/mL and 123.80 ± 13.5 pg/mL), and also result showed significant increase of mean sera level of IL-8 than TNF-α in formula feeding children comparative with breastfeeding children (187.87 ± 19.5 pg/mL and 119.93 ± 17.4 pg/mL, respectively). Conclusion: The finding of this study suggested that increased levels of IL-8 and TNF-α are present in all age groups, in male comparative with female, and also in pediatric diarrhea feeding by formula than breastfeeding and no differences of this cytokine according to consistency of diarrhea. These results contribute to using the immune profile as a serological marker for diagnosing diarrhea caused by E. coli O157:H7 in comparison with the control group.