Background: Pre-exposure prophylaxis (PrEP) is an important intervention for reducing the risk of HIV transmission among high-risk populations such as female sex workers in Africa, where HIV prevalence remains high. We aimed to assess the use of PrEP among female sex workers in Gulu, Uganda. Methods: In this community-based cross-sectional study, we included HIV-negative female sex workers purposely selected from hotspots within Gulu city, Uganda between February and March 2023. A semi-structured questionnaire was administered to collect data on sociodemographic characteristics, reproductive data, sexual practices, and self-reported PrEP use in the past 3 months. Symptoms of depression were screened using Patient Health Questionnaire-2 tool. Predictors of PrEP use was determined using modified Poisson regression analysis model. p < 0.05 was considered statistically significant. Results: We enrolled 273 female sex workers with a median age of 27 (interquartile range: 24–32) years. Overall, 181 (66.3%) participants used PrEP. PrEP use was associated with; regular source of income beside sex work [adjusted prevalence ratio (aPR): 3.7, 95% confidence interval (CI): 2.11–6.35, p < 0.001], being in a polygamous marriage (aPR: 6.9, 95% CI: 1.32–35.77, p = 0.022), practicing sex work in both rural and urban areas (aPR: 2.5, 95% CI: 1.49–4.35, p < 0.001), having symptoms of depression (aPR: 3.3, 95% CI: 1.43–7.74, p = 0.005), and use of postexposure prophylaxis (PEP) in the past 12 months (aPR: 0.31, 95% CI: 0.17–0.59, p < 0.001). Conclusion: Almost two in three of the female sex workers in Gulu city were currently using PrEP. Previous use of PEP was associated with lower use of PrEP. These findings suggest the need for targeted interventions to increase PrEP uptake and decrease HIV acquisition in this high-risk population among female sex workers, especially those with low income and limited access to healthcare.