Introduction HIV prevalence and sexual risk have been estimated very high for transgender people. However, the limited sampling and data collection methods used in current research on transgender people potentially led to overrepresentation and generalisation of people at risk for HIV. Current HIV prevalence estimates in transgender populations are generalised from studies mainly focusing on transgender women engaging in sex work. Moreover, studies focusing on non-binary people, who identify with a broad range of identities beyond the traditional male and female gender identities, are scarce. Objectives To estimate the HIV prevalence rate in the Flemish and Brussels (Belgium) transgender population, including transgender women, transgender men and non-binary people, and to identify the associated risk factors. Methods In this community-based cross-sectional study, self-identified transgender and non-binary (TGNB) people will be recruited through a two-stage time-location sampling approach. First, community settings in which TGNB people gather will be mapped to develop an accurate sampling frame. Secondly, a multistage sampling design is applied involving a stratification based on setting type (healthcare facilities vs outreach events), a selection of clusters by systematic sampling and a simple random selection of TGNB people within each cluster. Participants will complete an electronic self-reported survey to measure sociological, sexual and drug-using behaviors (risk factors) and oral fluid aliquots will be collected and tested for HIV antibodies. Logistic regression models will be used to evaluate risk factors independently associated with HIV infection. The presented study is registered at ClinicalTrials.gov (NCT04930614). Discussion This study will be the first to investigate the HIV prevalence rates and associated risk behaviors in an accurate representation of the TGNB population in a Western European country. The findings will globally serve as a knowledge base for identifying subgroups at risk for ...