Objective(s): Helicobacter pylori (H. pylori) is a gram-negative bacterium that plays a significant role in developing gastrointestinal diseases such as peptic ulcers. H. pylori infection affects more than half of the world’s population. This pathogen incidence becomes more effective among thalassemia patients, a common genetic disorder characterized by a point mutation that leads to impaired production of the β-globin chain. Chronic hepatitis C is a chronic disease caused by the hepatitis C virus (HCV) that has significantly increased morbidity and mortality rates in these patients due to liver failure or hepatocellular carcinoma. Methods: Current study included 90 β-thalassemia patients admitted to the thalassemia hematology center in Al-Kut women and children Hospital, Wasit province, Iraq from September 2020 to November 2021. The patients are divided into two groups (β-thalassemia with HCV group and β-thalassemia without HCV). First and second groups contain 32 and 58 specimens of blood and stool samples, respectively. The patient diagnosed with thalassemia syndrome regularly attended the thalassemia hematology center for transfusion and chelation and follow up on Hb level and iron status. This study aimed at serological and molecular diagnosis of co-infection H. pylori with Hepatitis C virus (HCV) among thalassemia patients and diagnosis infection rate. Diagnosis was used Serological enzyme-linked immunoassay (ELISA) techniques for HP-IgG, Level of ferritin, liver function test will be determined and Nested PCR method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool samples. Results: 90 β-thalassemia patients sample were diagnosed by Serological ELISA techniques for HP-IgG and the result showed 30 samples were positive (18 from the β-thalassemia with HCV group and 12 from the β-thalassemia without HCV)and 60 samples negative for HP-IgG and Nested polymerase chain reaction (PCR) method targeting the molecular diagnosis will be used to confirm H. pylori 23S rRNA using DNA extracted from stool for 30 stool samples revealed 13 samples positive (9 from the β-thalassemia with HCV and 4 from β-thalassemia without HCV) while 17 samples were negative. Conclusions: The predominance of H. pylori among patients in Wasit was 43.3%. Levels of ferritin have significant correlation with H. pylori in HCV patients. There was a positive correlation between hepatitis C status and high H. pylori rate when compared with β-thalassemia without HCV. Splenoctomy was a positive correlation with H. pylori in β-thalassemia with HCV. Moreover, these results recommend study about the virulence factor and genes. Other molecular studies are needed for detecting more virulence genes in H. pylori bacteria such as real-time PCR and sequencing of H. pylori in patients with thalassemia, especially those with high serum ferritin.