Aim: Personalized medicine (PM) is a novel approach to diagnose and treat disease. The study assessed the knowledge, attitudes and future expectations of healthcare professionals (HPs) towards PM in Ethiopia. Materials & methods: A cross-sectional survey with primary data and a simple random sampling technique was applied to collect data. Results: Our study revealed from a total of 384 respondents, 98 (25.5%), 146 (38%) and 140 (36.5%) had good, medium and poor knowledge of PM, respectively. However, 172 (44.8%), 185 (48.2%) and 27 (7%) had positive, neutral and negative attitudes towards PM, respectively. Conclusion: Most respondent’s future expectations of PM were positive. Education level had a significant association with attitudes and other sociodemographic variables were not significant for both knowledge and attitude.