A cross-sectional survey was conducted to investigate the nurses' knowledge of and compliance with Universal Precautions (UP) in an acute hospital in Hong Kong. A total of 450 nurses were randomly selected from a population of acute care nurses and 306 were successfully recruited in the study. The study revealed that the nurses' knowledge of UP was inadequate. In addition, UP was not only insufficiently and inappropriately applied, but also selectively practiced. Nearly all respondents knew that used needles should be disposed of in a sharps' box after injections. However, nurses had difficulty in distinguishing between deep body fluids and other general body secretions that are not considered infectious in UP. A high compliance was reported regarding hand-washing, disposal of needles and glove usage. However, the use of other protective wear such as masks and goggles was uncommon. The results also showed no significant relationships between the nurses' knowledge and compliance with UP. It is recommended that UP educational programmes need to consider attitudes in conjunction with empirical knowledge. Nurse managers and occupational health nurses should take a leadership role to ensure safe practices are used in the care of patients.