Fasciolosis, an infectious disease caused by the liver fluke Fasciola hepatica, affects grazing cattle world-wide. Liver fluke F. hepatica is prevalent and well-documented in cattle in many European countries, but for the Baltic countries such information is limited. This study investigated the seroprevalence and distribution of F. hepatica in cattle in Estonia. A total of 2461 individual serum samples from 218 farms distributed throughout all 15 Estonian counties, collected between February 2012 and March 2013, were tested for specific anti-F. hepatica antibodies using an in-house enzyme-linked immunosorbent assay (ELISA). In total, 144 individual animals tested seropositive, yielding an animal-level seroprevalence of 5.9% (95% CI 5.0–6.9). The herd-level seroprevalence was 28.4% (95% CI 22.8–34.7) and the herds with at least one seropositive animal were located in 13 of the 15 counties. Of the 62 F. hepatica-positive herds, 14 (6.4%) had an in-herd seroprevalence higher than 25%. With respect to production type, the herd-level seroprevalence was 20.2%, 35.6%, and 36.4% in dairy, mixed, and beef herds, respectively. Animals from the two large islands had higher odds of testing F. hepatica-seropositive than animals from the mainland. Animals from mixed and beef herds had higher odds of testing F. hepatica-seropositive than animals from dairy herds. Mixed and beef herds, and herds with more than 100 cattle, had higher odds of having at least one seropositive animal. This study provided the first serological evidence of the presence and distribution of F. hepatica in cattle herds in Estonia.