Background Family-Centered Care in Neonatal Intensive Care Units involves active family participation in the care of newborns and infants. Implementing family-centered care in neonatal intensive care units faces multiple challenges from the perspective of health care professionals. The neonatal intensive care environment can be stressful for parents, as health care professionals often prioritize the medical needs of newborns by addressing the emotional concerns and negative experiences of families. Objectives To assess the perception, practice and associated factors of family-centered care among health care professionals in the neonatal intensive care unit at Public Hospitals in Addis Ababa, Ethiopia, 2024. Method An institutional-based cross-sectional study was conducted in Neonatal Intensive Care Units (NICUs) of four randomly selected public hospitals using the lottery method. The study population included all health care professionals working in the NICU during the study period. The sample size was calculated using a single population proportion formula, and a total of 184 health care professionals were included using a survey sampling technique. Data was collected using the Family Centered Care Questionnaire-Revised (FCCQ-R). The collected data was coded and entered into SPSS version 27 for analysis. Descriptive statistics were used to summarize demographic characteristics and variable distributions, while bivariate and multivariable analyses were performed to identify associated factors. Result Out of the total 184 health care professionals, 57 (31%) had good perception and 67 (36.4%) had good practice of family-centered care. In multivariable logistic regression analysis, educational status of health care professionals (AOR = 0.237, 95% CI: 0.102–0.551) and negative staff attitude (AOR = 0.301, 95% CI: 0.127–0.712) were significantly associated with perception. Similarly, educational status (AOR = 0.359, 95% CI: 0.168–0.767) and negative staff attitude (AOR = 0.204, 95% CI: 0.086–0.482) were significantly associated with practice at p < 0.05. Conclusion and Recommendation: The practice of family-centered care among health care professionals was higher than their perception of family-centered care in the Neonatal Intensive Care Unit. Public hospitals should establish clear policies, guidelines, and accountability mechanisms to strengthen the implementation of family-centered care. In addition, health care providers should receive continuous education through both pre-service and in-service training.