Background: Cold chain management system still major challenge in developing countries including Ethiopia. Keeping vaccines has been one of the main quality assurances. This could mainly be defined by health professional’s knowledge and practices about on cold chain management ensure immunization potency. The aim of this study was to assess knowledge and practices of health care workers on cold chain management and associated factors in public health institutions. Objective: To assess knowledge and practices of health care workers on cold chain management and associated factors in public health institutions in Amhara region, South Gondar Zone, North-West Ethiopia, March 2020. Methods: An institution based cross-sectional study was conducted from March 11th– April 2nd/ 2020 in South Gondar Zone. Three hundred ninety six health workers were participated this study. Multistage systematic random sampling method was used to select the participants. Data was collected using structured self-administered questionnaire and observational checklist. Data were coded, cleaned and entered Epi data version 3.1 and exported to SPSS Version 25 and analyzed. Bivariable logistic regression P-value <0.25 was considered as cut of point to identify candidate variable; for multiple binary logistic regression P-value < 0.05 considered as statically significant. Result: Response rate was 98.7%. About 21.7%of respondents had good knowledge and 11.5% had good practice on cold chain management. The mean (±SD) age of respondents was 27.8 ±3.9years.Regarding to their profession 51.7%, 17.4%, 15.9%, 15.1% of respondents’ were nurses, midwifery, public health officer and pharmacy respectively. Trained on vaccine cold chain management (AOR=12.28; 95% CI=6.33-23.28 ;) and availability of guideline in health facility (AOR= 2.17; 95% CI=1.11-4.22 ;) had given good knowledge on cold chain management. Likewise, being urban residence (AOR=2.45; 95% CI=1.28-4.70) and avail guideline (AOR=2.55; 95% CI=1.33-4.92) had given good practice on cold chain management. Conclusion: Health professionals working in public health facilities and had low knowledge and practice on cold chain management. Training, availability guidelines and working urban at work were factors that improved health professionals’ knowledge and practice about cold chain management, which needs to be sustained. Keywords: Cold chain management, Vaccine, Practice and Knowledge.