Background: Health workers turnover is an increasing problem that threatens the functioning of the health care sector worldwide, especially in developing countries. There are very few studies on turnover intention in low-income countries, especially in Ethiopia. This study aimed to assess turnover intention of health workers and its determinants in University of Gondar Referral Hospital, Ethiopia. Methods: Institutional based cross sectional study was conducted from March to April, 2014 at University of Gondar Referral Hospital on 394 health professionals using stratified random sampling techniques. The data were cleaned, coded, entered into EPI INFO version 3.5.3, and transferred and analyzed using SPSS version 20. Bi-variable and multivariable logistic regression analyses were carried out to identify factors associated with turnover intention. Results: overall, 52.5% of the health professionals reported to have turnover intentions. Respondents who have degree and above academic rank [AOR=2.717(95%CI:1.192,6.190)]; nursing profession [AOR= 7.668(95%CI: 2.913,20.188)], laboratory profession[AOR 9.153,(95% CI: 2.925,28.636)], work experience of 2.1 to 5 years [AOR=1.937,(95% CI: 1.142,3.288)] and income level of 2100 to 2259 [AOR =0.431(95% CI: 0.213,0.871)] were significantly associated with turnover intention of the health professionals. Conclusions: In this study, turn over intention of health professionals was found to be high. Educational level, profession, work experience, income are significantly associated with intent to turnover. Hence, it is worth strengthening human resources for health management at hospital level through implementing retention strategies.