The study investigated the sero-status of human immunodeficiency virus among healthcare workers in Addis Ababa public hospitals. A multi-centered, institutional-based, cross-sectional study was conducted from 18 September 2022 to 30 October 2022. A simple random sampling method and a semi-structured, self-administered questionnaire were used to collect the data, which were analyzed using the Statistical Package for Social Sciences (SPSS) version 25. A binary logistic regression model was used to identify the factors associated with the human immunodeficiency virus sero-status of healthcare workers post exposure to infected blood and body fluids. Of the 420 study participants who were exposed to blood and body fluids, 403 (96%) were non-reactive. Healthcare workers who had 20–29 years of work experience had approximately six times higher odds of testing positive for the human immunodeficiency virus (AOR = 6.21, 95% CI: 2.39, 9.55). Healthcare workers who did not use personal protective equipment properly had five times higher odds of testing positive for the human immunodeficiency virus (AOR = 5.02, CI: 3.73, 9.51). This study showed that, among those healthcare workers who tested positive for the human immunodeficiency virus infection, the majority were from the emergency department. Healthcare workers who did not use personal protective equipment properly had higher odds of testing positive for the human immunodeficiency virus.