Tick-borne encephalitis virus (TBEV) is an arbovirus that causes severe infections in humans, and is endemic to large areas of Europe and Asia. Humans most commonly become infected with TBEV after a tick bite; however, alimentary infection can occur after consumption of unpasteurized dairy products. Milk from sheep and goats can be a source of alimentary TBE infections. In addition, sheep and goats are considered suitable sentinels for surveillance of TBEV-associated risks in endemic areas. Here we conducted a serological survey to determine the prevalence of TBEV infection among sheep and goats in the Czech Republic. In 2019–2020, a total of 310 serum samples were collected from sheep and 418 from goats, in 11 of the 14 administrative districts of the country. Sera were tested for the presence of TBEV-specific IgG by ELISA, and suspected results were validated using a virus neutralization test. Positive samples were identified in 56.7% of goat farms, and 82.4% of sheep farms, and in 9 of the 11 administrative districts examined. The seroprevalence was significantly higher among sheep (32.5%) than goats (19.7%) (p < 0.001). The present results indicate that sheep and goats have a relatively high rate of exposure to TBEV-infected ticks in most of the administrative districts of the Czech Republic. These findings confirm the usefulness of serological testing in small ruminants to determine and monitor the risk of TBEV infection in humans.