Back ground: Human immunodeficiency virus infection is a severe public health issue that claims the lives of numerous people, including medical professionals. The effectiveness of daily oral pre-exposure prophylaxis for HIV prevention among at-risk individuals has thus been repeatedly shown in clinical trials. An important public health opportunity to reduce HIV incidence is presented by pre-exposure prophylaxis. Objective: The purpose of this study is to evaluate health care workers' knowledge, attitudes, and factors related to pre-exposure prophylaxis against HIV/AIDS at Debretabor comprehensive specialized hospital in Debretabor, Ethiopia. Method: A cross-sectional study was conducted among 322 health care workers in Debretabor comprehensive specialized hospital from June to July, 2023. The data were collected by trained data collectors using a structured questionnaire with self-administered questioners. The collected data were entered, and the data were checked, coded, data cleaning and analysis using SPSS version 23. Result: The level of attitudes of pre-exposure prophylaxis was analyzed among all 322 respondents. Overall attitudes of health care workers were 68% [95% CL: 62.7-72.7].Off 322 study participants were participated with a response rate of 100%. About 134(41.6%) health care workers had six to ten year clinical experience in their profession, and almost half, 63(51.6%) health care workers in this study were Nurses followed by 62(19.3%) doctors. Majority of 203(63%), and 127(39.4%) were male, and educational status were BSc degree, Prior to conduct this survey, 273 (84.8%) the respondents were aware of the concept of HIV pre-exposure prophylaxis. but only half of, 171 (53.1%) Previously took training on pre-exposure prophylaxis. And 37 (11.5%) of participants had self-rating poor knowledge about HIV- pre-exposure prophylaxis. Variables which had P value less than 0.25 in bivariable analysis would be adequate for multivariable logistic regression analysis, and then in the final stage of multivariable analysis two variables such as marital status [AOR:.211(0.048-0.932],educational status[AOR:.032(.003- .365],[AOR:.181(0.054-0.600)],[AOR:.112(0.027-0.469)],[AOR: .084(0.025-0.286)], and [AOR: .204(0.058-0.720)] of health care workers had significantly associated with the attitudes of pre-exposure prophylaxis.. Conclusion and Recommendation: Our study shows that, inadequate knowledge, and attitude towards pre-exposure prophylaxis, and its management comparing to previously conducted researches, but the current study shows low levels of attitudes, there is suboptimal knowledge about PrEP. So concerned bodies expected to give training to health care workers to increase knowledge, and attitude of health care workers, and researchers expected to conduct research in the area of pre-exposure prophylaxis.