Background : Infection is one of the diseases in Indonesia caused by the lack of knowledge to perform Hand Hygiene. Hand Hygiene is hand cleaning action appropriately and correctly by doing hand wash and handrub. Hand wash is wash their hands using soap and water flows. Hand Rub is clean hands using alcohol-based fluid. Hand Hygiene is the right way in order to prevention of infection. Purpose :ResearchThe Relationship Between Education, knowledge and the ability to Hand Hygiene with the incidence of disease at Madrasah students Mu'allimaat Muhammadiyah Yogyakarta. Metode :This research is Analytically Quantitative With the approach of Cross Sectional. The population of this research amounted to 79 respondents, 40 High Schools and 39 Junior Secondary Schools. The sampling technique used Proportionate Simple Random Sampling method in Madrasah Mu’allimat Muhammadiyah Yogyakarta on April - September 2016. Data was collected by distributing questionnaires using Chi-square statistical test. Result :Relation between education and the incidence of disease0.035 (<0.05) weak category, Hand Hygiene knowledge relationship with the incidence of disease 0.000 (<0.05) strong category and the ability of Hand Hygiene in the incidence of disease 0.038 (<0.05) weak category. Conclusions:There is a relationship between education, knowledge and the ability to Hand Hygiene with the incidence of disease at Madrasah students Mu'allimaat Muhammadiyah Yogyakarta. It is necessary for the socialization of the school to increase awareness of students to optimize the ability to perform Hand Hygiene.