Background: Depression is the world’s leading cause of disability and a major contributor to the overall global burden of disease. Female sex workers operating in conflict-affected settings could be more prone to depression, yet the prevalence and factors associated with depression among this special population remain inadequately addressed. This study thus aimed to determine the prevalence and factors associated with major depression among female sex workers in post-conflict Gulu district. Methods: This cross-sectional study was conducted among 300 randomly selected female sex workers in Gulu district between March and June 2020. Possible factors associated with depression were assessed using pre-tested semi-structured questionnaire and participants assessed for major depression using Mini-International Neuropsychiatric Interview (MINI) version 7.0.0. We utilized STATA 14.0 for univariate, bivariate and multivariate data analyses.Results: The mean age of study participants was 26.4 years (SD = 6), 57.7% attained primary education, 51.7% never married and 42.1% were living with HIV. Almost half (47.7%) of the participants had major depression, of whom half (50.4%) had severe depression and one-thirds (34.3%) had suicidal ideation. At multivariate level, factors that showed positive association with major depression were: psychological stressor (aOR = 11.0, 95%CI: 5.77-20.86), living with HIV (aOR= 2.32, 95%CI: 1.29-4.15), being verbally abused (aOR = 2.27, 95%CI: 1.26-4.07), and being 30+ years of age (aOR = 1.95, 95%CI: 1.05-3.61). Meanwhile, factors that showed negative association with major depression were: providing sexual services at client’s place (aOR=0.46, 95%CI: 0.24-0.91), using modern family planning method (aOR=0.47, 95%CI: 0.25-0.87), and daily intake of alcohol (aOR=0.50, 95%CI: 0.28-0.88).Conclusion: The findings underscore the high prevalence of major depression among female sex workers driven by psychological stress, living with HIV/AIDs, being verbally abused by clients and being 30+years of age. The high prevalence of major depression has important public health ramifications at individual and societal levels and requires urgent attention of all stakeholders.