Purpose To assess health-related quality of life (HRQoL) and its associated factors among people living with HIV/AIDS (PLWHA) in Rio de Janeiro, Brazil. Methods A cross-sectional study including PLWHA receiving usual HIV-care at Instituto Nacional de Infectologia Evandro Chagas (INI/Fiocruz) was conducted between 2014 and 2016 in Rio de Janeiro, Brazil. The EQ-5D-3L assessed HRQoL; PHQ-2 and ASSIST were used for screening depression and substance use, respectively. Clinical variables were obtained from the INI/Fiocruz cohort database, and structured questions evaluated intimate partner violence, sexual abstinence and relationship status. Data were analysed using multivariable Tobit regression model. Results A total of 1480 PLWHA were included: 64.7% were male at birth (38.4% men who have sex with men [MSM], 24.3% heterosexual men and 2% transgender women [TGW]); median age was 43.1 years, and 95.8% were receiving antiretroviral therapy. The median EQ-5D-3L utility score was 0.801. Results showed that the following factors: MSM and women; older age; lower educational level; no engagement in a relationship; depression screening positive; polysubstance use; and, detectable viral load were independently associated with worse HRQoL. Conclusions PLWHA under care at INI/Fiocruz presented good HRQoL. Polysubstance use, depression and lower educational level were among the factors negatively associated with HRQoL. This was the first time that the EQ-5D-3L utility scores were calculated for a considerable number of PLWHA in Brazil, which is a fundamental piece of information for future cost-effectiveness analysis.