Ultra-processed food is one of the main contributors to energy supply and consumption in food systems worldwide, and evidence of their detrimental health outcomes in humans is emerging. This study aimed to assess ultra-processed food intake and its association with urinary levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of DNA oxidative damage, in 139 healthy adolescents in Karaj City in Iran. Usual dietary intake was measured using a 168-item validated FFQ. The daily intake of ultra-processed food consumption was determined through the classification of NOVA, and general linear models were used to compare the urinary levels of 8-OHdG/creatinine (ng/mg creatinine) within tertiles of ultra-processed food intake. Adolescents in the higher tertile of ultra-processed food consumption had a significantly higher mean level of urinary 8-OHdG/creatinine in comparison with the lower tertiles in the crude model (Pfor trend: 0·003) and after adjustment for confounding variables, including total energy intake, sex, age, BMI for age Z-score, obesity and physical activity (Pfor trend: 0·004). This association was still significant after adjusting for dietary intake of whole grains, nuts, legumes, the ratio of MUFA:SFA (g/d) and Mediterranean dietary score (Pfor trend: 0·002). More studies are needed to explore the determinants of ultra-processed food supply, demand, consumption and health effects; such studies should be applied to develop evidence-informed policies and regulatory mechanisms to improve children’s and adolescents’ food environment policymaking and legislation with special attention to ultra-processed food.