Nosocomial infections are infections that are spread through hand contact and risk to the health care system in the hospital. One way that is most convenient and effective to prevent nosocomial infections is hand hygiene. Health workers in the hospital were most responsible in this case is a nurse. Many factors affect the ability of hand hygiene in nursing, among which education and knowledge about hand hygiene. The purpose of this study was to determine the relationship of education and knowledge with the ability of hand hygiene in nursing. This research is an analytic observational study with cross sectional approach. This research was conducted in hospitals Karanganyar with the number of respondents as many as 30 nurses. Sampling technique is using proportionate random sampling. The results showed no significant association between level of education and knowledge of hand hygiene (p = 0.923), there wasn’t a significant relationship between the level of education with the ability of hand hygiene (p = 0.560), there wasn’t a significant relationship between the level of knowledge and ability with the ability of hand hygiene (p = 0.104) in Karanganyar District Hospital. Socialization is required from the hospital to increase self-awareness of each nurse to further optimize the ability to perform hand hygiene based on the education level of its.