Non-alcoholic fatty liver disease (NAFLD) is a relatively common disease, and preventing its occurrence is important for both individual health and reducing social costs. Shift work is reported to have several negative effects on health. An association has been observed between NAFLD and both sleep time and quality; however, this association remains unclear in night shift workers. We aimed to evaluate the relationship between shift work and the incidence of NAFLD. Overall, 45,149 Korean workers without NAFLD were included at baseline. NAFLD was defined as the presence of a fatty liver observed on ultrasonography without excessive alcohol use. incidence rate ratios for incident NAFLD were estimated using negative binomial regression according to age groups (20s, 30s, 40s, and 50s). In the 20s age group, shift work showed a significant incidence rate ratio (IRR) for NAFLD in all models. After adjusting for all variables, the IRR (95% confidence interval) was 1.24 (1.08–1.43) in the 20s age group. In their 20s, a significant association between shift work and incident NAFLD was consistently observed among women and workers with poor sleep quality. In this large-scale cohort study, shift work was significantly associated with the development of NAFLD among young workers in their 20s.