Objective: To assess the knowledge of healthcare workers on the measles vaccine and its cold chain management. Method: An institutional-based cross-sectional study was conducted from February 1 to March 30, 2022 in Gondar City Administration public health institutions among 165 healthcare workers. Data were collected using a structured questionnaire. In addition, an on-spot observation checklist was used to assess the availability, status and management of the cold chain. A logistic regression model was used to assess the relationship between the outcome and predictor variables. Crude and adjusted odds ratios were calculated with 95% confidence intervals. Results: Overall, 87 (52.7%; 95% Cl 44.8%-60.5%) of the healthcare workers had unsatisfactory knowledge regarding the measles vaccine and its cold chain management. One hundred thirty-six (82.4%) healthcare workers correctly mentioned the recommended range of temperature (2-8 °C) for measles vaccine storage. Healthcare workers aged 18-29 years (P=0.001) and 30-44 years (P=0.014) were observed as determinants of unsatisfactory knowledge on the measles vaccine and its cold chain management. One hundred and five (63.6%) of the healthcare workers did not correctly mention the type of measles vaccine used in routine immunization. More than one-third (36.4%) of the healthcare workers perceived that the measles vaccine is not safe and could cause measles. Conclusions: More than half of the healthcare workers in the study area had unsatisfactory knowledge on the measles vaccine and its cold chain management. It is necessary to provide technical support and in-service training for healthcare workers to ensure optimal immunization effectiveness.