Hepatitis E virus (HEV) is a major cause of acute hepatitis in humans, and strains of genotypes 1 and 2 are endemic in many regions with suboptimal sanitary conditions. In many industrialized countries, HEV strains of genotype 3 are highly endemic in swine, and an increased number of autochthonous infections with HEV genotype 3 strains have been reported lately. Serological studies of HEV infection are often conducted with commercial assays based on peptides and recombinant proteins of HEV genotype 1 and 2 strains. For some patients with proven HEV genotype 3 infections, these assays failed to detect specific antibodies, and they are not applicable or validated for the detection of anti-HEV antibodies in swine. To elucidate the incidence of hepatitis E in regions where HEV genotype 3 infections can be expected, and to study the seroprevalence of HEV in swine, new tools with broad specificity for all genotypes of HEV are needed. We present the expression and partial characterization of recombinant HEV genotype 3 open reading frame 2 (ORF-2) proteins and their usefulness as diagnostic antigens in detecting anti-HEV antibodies in humans and swine with proven HEV genotype 3 infections. The recombinant antigens were produced at relatively high yields and at low cost upon infection of Trichoplusia ni larvae with recombinant baculoviruses expressing recombinant HEV genotype 3 ORF-2 proteins. The enzyme-linked immunosorbent assay based on the recombinant proteins showed good specificity and sensitivity for anti-HEV genotype 3 immunoglobulin G detection in human and swine sera. These recombinant HEV genotype 3 ORF-2 proteins might be added to diagnostic kits containing HEV genotype 1 and 2 antigens in order to develop a broadly sensitive new tool for the diagnosis of hepatitis E.