Background: Protecting the sleep of premature infants is crucial, particularly in the neonatal intensive care unit (NICU). Health care professionals in NICUs play a vital role in supporting and ensuring the protection of infants' sleep. Aim: The study aimed to examine the knowledge, attitudes, practices and perceived barriers among NICU health care professionals in Türkiye regarding the protection of premature infants' sleep. Study Design: This cross-sectional study was conducted online through social media platforms, involving 104 health care professionals (physicians and nurses). A survey was used to evaluate their knowledge, attitudes, practices and perceived barriers in protecting the sleep of premature infants. Results: The total scores of NICU health care professionals in knowledge, attitude, practice and perceived barriers were as follows: knowledge score, 27.10 ± 2.126; attitude score, 8.65 ± 0.942; practice score, 21.25 ± 2.139; and perceived barriers score, 26.98 ± 2.028. Significant relationships were found between knowledge and attitude (p =.003), knowledge and practices (p =.005), attitude and practices (p <.001), attitude and perceived barriers (p <.001) and practices and perceived barriers (p =.005). Conclusion: NICU health care professionals demonstrated high levels of knowledge, attitude, practice and perceived barriers regarding the protection of premature infants' sleep. A positive relationship was observed between knowledge and attitude, knowledge and practices, and attitude and practices. Conversely, a negative relationship was found between attitude and perceived barriers, as well as practices and perceived barriers. Relevance to Clinical Practice: It will be easier to protect the sleep of premature infants by determining the knowledge, attitude, practice and perceived barriers of NICU health care professionals. In particular, by examining the knowledge, attitudes and practices of nurses regarding sleep protection and identifying the obstacles to successful sleep management, premature infants' sleep will be of better quality.