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Background: Poor sleep quality is linked to physiological dysfunction, which increases the risk of obesity, cardiovascular disease, cognitive impairment, and other medical conditions. Despite the known health risks of sleep disturbances, literature is still scant regarding sleep quality and its associated factors among healthcare professionals in Ethiopia. Therefore, this study aimed to determine the prevalence of poor sleep quality and its associated factors among healthcare professionals at the University of Gondar Comprehensive Specialized Hospital. Methods: An institution-based cross-sectional study was conducted at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. A total of 418 healthcare professionals participated in the study. The study participants were chosen using the stratified random sampling method. Data were collected using a structured, self-administered questionnaire. The Pittsburgh Sleep Quality Index (PSQI) was used to assess the sleep quality of participants. Bi-variable and multivariable logistic regression models were used. p ≤ 0.05 was used to declare statistically significant variables. Results: The mean age of the study participants was 30.7 years (SD ± 6.3). The overall prevalence of poor sleep quality was 58.9% [95% CI (54.2, 63.6%)]. Being female [adjusted odds ratio (AOR) = 1.9, 95% CI (1.2, 2.9)], being a shift worker [AOR = 5.7, 95% CI (2.3, 14.3), not performing regular exercise [AOR = 2.08 (1.2-3.6)], being a khat chewer [AOR = 3.1, 95% CI (1.2, 7.6)], and having depressive symptoms [AOR = 2.6, 95% CI (1.3, 6.8)] were significantly associated with higher odds of having poor sleep quality. Conclusion: The prevalence of poor sleep quality among healthcare professionals was found to be high. As a result, we recommend that healthcare providers at the University of Gondar Comprehensive Specialized Hospital focus on early regular screening for sleep disturbances and pay special attention to shift work schedules and behaviors such as khat chewing, exercise, and depressive symptoms. [ABSTRACT FROM AUTHOR]