Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), poses significant challenges to the global livestock industry, particularly affecting bovine populations. To better understand the prevalence of paratuberculosis and its diagnostic nuances, a comprehensive meta-analysis was conducted. This analysis encompassed 21 studies involving 632,767 cows for milk enzyme-linked immunosorbent assay (ELISA) and 51 studies involving 256,409 cows for serum ELISA. The pooled prevalence estimate for paratuberculosis on a cow-basis was found to be 16% (95% CI: 14%; 18%) for milk ELISA and 8% (95% CI: 7%; 8%) for serum ELISA. Notably, higher confidence intervals (CI) were observed in milk ELISA, the Europe and Asia groups, suggesting variability in prevalence estimates within these regions. Conversely, lower CIs were noted in the USA and Canada groups, indicating greater consistency in prevalence estimates within these countries. However, serum ELISA exhibited high CI values across all regions, underscoring potential variability in diagnostic performance. These findings provide valuable insights for veterinarians, researchers, policymakers, and livestock producers in optimizing paratuberculosis detection and control strategies to mitigate its impact on bovine health and agricultural productivity.