Stray animals are unowned free roaming, homeless, abandoned, street or sheltered animals, particularly dogs, cats and cattle. They could act as carrier of several zoonotic pathogens such as rabies virus, Mycobacterium and Brucella species. However, comprehensive information on the prevalence of zoonotic pathogens in stray animals is very limited. We conducted a systematic review as per Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) 2020 guidelines to estimate the prevalence of brucellosis in stray dogs, cats and cattle. Eligibility criteria for the study were determined using the PECOS classification (population, exposure, comparison, outcome, study design) as a tool to guide the research and adjust the search strategy. Major bibliographic databases [Web of Science, Medline, Scopus, ScienceDirect, Google Scholar and PubMed] were searched using predefined keywords for published epidemiological studies on brucellosis in stray animals (dogs, cats and cattle). Systematic assessments of all the studies since 1990-2022 were conducted and those reporting the prevalence of brucellosis in stray dogs, cats and cattle using appropriate diagnostic tests (culture, molecular, serological) were included. Studies reporting prevalence of brucellosis (Brucella infection or exposure) in kennel dogs, dairy herds, livestock farms, humans or marine species were excluded. The apparent individual test- wise prevalence along with 95% confidence intervals (CI) was estimated using Epitools. Out of 2689 studies, 37 met the inclusion criteria and were included in the systematic review. Of 37 studies, 28 (75.7%) were conducted in stray dogs, 7 (18.9%) in cattle and 2 (5.4%) in cats. Furthermore, only 21.62% studies (8/37) used probabilistic random sampling approaches and 13.51% studies (5/37) explained and justified the study sample size using appropriate methods for estimation of disease prevalence in the study populations. Higher sero-prevalence in stray dogs has been reported in studies conducted in Jordan (38.0% (95% CI: 24.0-54.0) and Pakistan (38.0% (95% CI: 31.0-45.0) whereas no sero-positivity was recorded in the studies conducted in Brazil, North Colombia, Cyprus, South Korea and USA. All studies on brucellosis (n = 7) in stray cattle were from India; conducted in stray cattle reared in cow-shelters. Sero-prevalence in the range of 4.3%- 64.3% was reported in stray cattle. Differences in diagnostic tests and host species, as well as limited number and non-randomized studies and high statistical heterogeneity did not allow us to determine combined meta-analysed prevalence estimates. Stray animals are likely to pose a zoonotic and disease spillover risk to human and livestock populations.