Purpose: Staphylococcus aureus carriage poses an increased risk of S. aureus infection. The aim of this study was to investigate the colonization of S. aureus among healthy individuals and to establish a prospective cohort and biobank for research in the health consequences of colonization. Population and methods: The Danish Blood Donor S. aureus Carriage Study (DBDSaCS) was established in 2014. So far, a total of 6082 healthy participants have been included with nasal swabs and repeated swabs are performed at subsequent donations. Samples from the first 2217 participants were cultured using a two-step method to evaluate the effect of using enrichment broth. Furthermore, 262 participants were sampled from both the nares and the throat. All participants completed a questionnaire with self-reported health, anthropometric measurements, current smoking status, and physical activity. Plasma samples, nasal swab transport media, and S. aureus isolates were stored. Results: The prevalence of S. aureus nasal colonization was 41%. The prevalence of colonization was higher in men (46%) than women (34%), lower for smokers, and decreased with increasing age (<25 years: 44% vs >55 years: 35%). In participants swabbed from the nose and throat, the prevalence of S. aureus colonization after enrichment was 55% with significantly higher prevalence in the throat (45%) than in the nose (40%). The use of an enrichment broth increased the proportion of S. aureus colonization. Conclusion: We describe a large and growing cohort of healthy individuals established to investigate predictors for S. aureus carriage and the health consequences of carriage. Multiple projects using data from DBDSaCS linked with Danish health registers, biomarkers, and genetic markers are ongoing. Results will be published in the coming years.