Introduction: Biomedical waste management is the process of segregation, storage, treatment, disposal and other safety measures of waste in the health institutions and it is an important precondition to safe guard the healthcare workers, community and environment from being contaminated with infectious substances related to biomedical waste. However, biomedical waste management is a problem in most developing countries and biomedical waste is an ongoing problem in health care facilities and poses a serious public health problem. Objective: To assess biomedical waste management practice and associated factors among health care workers at metropolitan city private hospitals, Amhara Region, Ethiopia. Methods: Institutional based cross-sectional study was conducted at metropolitan cities private hospitals in Amhara Region. By using simple random sampling 440 healthcare workers was selected. Data were collected using Self-administered questionnaire and observational check list. Training was given to data collectors and supervisors. The data were entered into Epi data version 4.6 and analyzed using SPSS version 20. To identify factors associated with the dependent variable, bi-variable logistic regression was used and then all variables with p-value < 0.25 were entered in to a multivariable logistic regression model for further analysis and Model fitness was checked by the Hosmer Lemshow goodness of fit test. Finally variables with p value <0.05 were considered as significantly associated at 95% CI. Result: Less than half of (49.4%) of health care workers had good practice of biomedical waste management. This study found that Health care workers who hold education level MSc and above [AOR=4.20, 95% CI :( 1.013, 17.40)], who attended degree [AOR=3.52, 95% CI :( 2.13, 5.82)],who took training [AOR=4.33, 95% CI :( 2.709, 6.929)], who had three bins in their working department [AOR=6.24.95% CI :( 3.840, 10.131)] and who had good attitude (AOR=2.640, 95% CI :( 1.649, 4.215) were practiced good biomedical waste management than their counterparts. Conclusion: Biomedical waste management practice was found low among health care workers. Different factors affect this practice such as Level of education, taking training on biomedical waste management, availability of three bins and attitude of health care workers were identified. Therefore, all responsible bodies should take action based on the identified factors. Keywords: biomedical waste, biomedical waste management, healthcare wo