The current burden of Hepatitis C virus infection and the availability of HCV-related services in Ghana are not well described. Previous estimates on HCV seroprevalence in the country are outdated. This study investigated the HCV seroprevalence and testing and treatment capacity in Ghana. A multi-centre cross-sectional study was conducted in which laboratory and blood bank registers from 17 public healthcare institutions in Ghana were reviewed. A survey on cost and availability of HCV-related testing and treatment was also performed. Crude and pooled estimates of HCV seroprevalence, frequency and median cost of available diagnostic tests and medicines were described. The crude HCV seroprevalence was 2.62% (95% CI 2.53–2.72) and the pooled estimate was 4.58% (95% CI 4.06–5.11) among 103,609 persons tested in laboratories. Age (OR 1.02 95% CI 1.01–1.02) and male sex (OR 1.26 95% CI 1.08–1.48) were predictors of a positive anti-HCV RDT test. Northern administrative regions in Ghana had the highest HCV seroprevalence ranging from 8.3–14.4%. Among 55, 458 potential blood donors, crude HCV seroprevalence was 3.57% (95% CI 3.42–3.72). Testing was through Rapid Diagnostic Test (RDT) kits in most facilities, and only 2 of 17 centres were performing HCV RNA testing. The median cost of an anti-HCV RDT test was $0.97 (0–1.61) and $3.23 (1.61–7.58) for persons with and without government health insurance respectively. The median cost of a 12-week course of the pan-genotypic direct-acting antiviral therapy sofosbuvir-daclatasvir was $887.70. In conclusion, there are significant regional differences in HCV burden across Ghana. Limited access to and cost of HCV RNA and DAA therapy hinders testing and treatment capability, and consequently HCV elimination efforts. A national HCV program supported with a sustainable financing plan is required to accelerate HCV elimination in Ghana.