Safety practice is an important element of workplace safety and quality of health care. To investigate the safety practice and professional exposure to blood and blood-containing materials during a one-year period among Health Care Workers (HCWs) in Serbia. Cross-sectional study of 1559 Serbian HCWs using a self-administered questionnaire. Mantel-Haenszel statistics and multiple logistic regression analysis were used in statistical analysis. Fifty-nine percent (921) of HCWs had skin contact with patients blood, followed by 51% (791) with needle stick injuries, 38% (599) with cuts from sharp instruments, and 34% with contact of eye and other mucosa with patient's blood. Nurses reported professional exposure more often than others. Safety practices consisted of using appropriate barriers (gloves, mask, glasses) in all procedures with patients and were used by 58%, 23%, and 4% of HCWs, respectively. Doctors protected themselves more regularly than others. Hospital protocols for post exposure prophylaxis and safety disposal of medical waste are not common in Serbian health care settings. Safety practices in use were having hospital guidelines for safety practice in hospitals [odds ratio (OR)=1.58, 95% confidence interval (CI)=1.14-2.19], carrying out some form of intervention with risks of infection (OR=3.76, 95% CI=2.57-5.51), and HCWs aware of the professional risk of acquiring infection (OR=1.48, 95% CI=1.28-1.79). This study indicates that emphasis on work practice, attire, disposal systems and education strategies, should be employed to reduce professional exposure to blood and blood containing materials among HCWs in Serbia.