Introduction: Bovine herpesvirus 6 (BoHV6) belongs to the Herpesviridae family, Gammaherpesvirinae subfamily and Macavirus genus. It is common in cattle, but was also detected in American bison (Bison bison) and water buffalo (Bubalus bubalis). The aim of the experiment was to develop an ELISA for serological examination of cattle sera for the presence of anti-BoHV6 specific antibodies. Material and methods: Viral DNA from a BoHV6-positive cow was amplified by qPCR and the resulting fragments of the gB and gH genes encoding glycoproteins B and H (gB and gH) were cloned into the pLATE52 vector to express recombinant gB (rgB) and gH (rgH) in Rosetta (DE3) E. coli. The expressed recombinant proteins were used as antigens in the developed ELISA. Results: The proteins expressed had the expected molecular weight. A total of 143 sera were examined, and 141 of them were positive, according to the chosen cut-off values of 9% and 10% for the sample-to-positive ratios of the rgB and rgH antigens, respectively. Conclusion: The rgB and rgH recombinant antigens of BoHV6 were successfully expressed in E. coli and successfully used in a newly developed ELISA.