Abstract Background This paper outlines the methodology, study population and response rate of a third large Dutch population-based cross-sectional serosurvey carried-out in 2016/2017, primarily aiming to obtain insight into age-specific seroprevalence of vaccine-preventable diseases to evaluate the National Immunization Programme (NIP). In addition, Caribbean Netherlands (CN) was included, which enables additional research into tropical pathogens. Methods A two-stage cluster sampling technique was used to draw a sample of Dutch residents (0–89 years) (NS), including an oversampling of non-Western migrants, persons living in low vaccination coverage (LVC) areas, and an extra sample of persons born in Suriname, Aruba and the former Dutch Antilles (SAN). A separate sample was drawn for each Caribbean island. At the consultation hours, questionnaires, blood samples, oro- and nasopharyngeal swabs, faeces, − and only in the Netherlands (NL) saliva and a diary about contact patterns – were obtained from participants. Vaccination- and medical history was retrieved, and in CN anthropometric measurements were taken. Results In total, blood samples and questionnaires were collected from 9415 persons: 5745 (14.4%) in the NS (including the non-Western migrants), 1354 (19.8%) in LVC areas, 501 (6.9%) SAN, and 1815 (23.4%) in CN. Conclusions This study will give insight into protection of the population against infectious diseases included in the NIP. Research based on this large biobank will contribute to public health (policy) in NL and CN, e.g., regarding outbreak management and emerging pathogens. Further, we will be able to extend our knowledge on infectious diseases and its changing dynamics by linking serological data to results from additional materials collected, environmental- and pharmacological data.