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 food frequency questionnaire. 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 crn) 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 to the lower tertiles in the crude model (P for trend: 0.003) and after adjustment for confounding variables, including total energy intake, sex, age, body mass index (BMI) for age Z-score, obesity, and physical activity (P for trend:0.004). This association was still significant after adjusting for dietary intake of whole grains, nuts, legumes, the ratio of monosaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) (g/day), and Mediterranean dietary score (P for 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 policy-making and legislation with special attention to ultra-processed food.