Background: Hepatitis B (HBV) Infection cause major public health problem around the globe including Ethiopia. Therefore, the aim of this study was to assess sero-prevalence of HBV marker (HBsAg, anti-HBc and anti-HBs) and associated factors among 5-8 years old children at Hawassa City, Southern Ethiopia. Methods: A community-based cross-sectional study was conducted in Hawassa city southern Ethiopia from September to May, 2018. A total of 469 children were included in the study by multistage sampling technique. Data on sociodemographic variable and associated factors was collected by using structured and pre-tested questionnaire. Blood sample was collected and sera was screened for hepatitis B surface antigen (HBsAg), Antibody to core antigen (Anti-HBc), and Antibodies against surface antigen (Anti-HBs) using ELISA. Data were analyzed using SPSS version 23. Results: The sero-prevalence of HBsAg, Anti-HBc, and anti-HBs among children was 4.4% [95% confidence interval (CI): 2.8–6.6], 19.5% [95% confidence interval (CI): 16.1–23.4] and 20.0% [95% confidence interval (CI): 16.5–23.8], respectively. Children who had history of injectable medications (AOR 5.02: 95% CI: 1.14, 22.07), family history of liver disease (AOR 6.37, 95% CI: 1.32, 30.74)), mothers who had HBsAg in their sera (AOR 11.19, (95% CI: 3.15, 39.67) and had no history of vaccination (AOR, 6.3795% CI: 1.32, 30.74) were more likely to had hepatitis B virus infection. High seropositivity of anti-HBc was detected among children from the family earn low monthly income, home delivered, no history of HBV vaccination and seropositivity mother for HBsAg had significant association with anti-HBc. Conclusions: The prevalence of HBsAg among children in Hawassa city was intermediate and endemic in prevalence. History of injectable medication, family history of liver disease, had no history of vaccination and maternal HBsAg seropositivity were independent risk factor for HBV infection. Therefore; this evidence suggests strengthening immunization coverage, safe injection practice and introducing screening program during pregnancy as part of the care xii package during antenatal care visits. Programs focusing on primary prevention of HBV infection in women and children could also be strengthened.