Human adenovirus 3 (HAdV-3) occurs epidemically in China in recent years. It can cause life-threatening infection in young children and immunocompromised patients. Development of reliable diagnostic reagents for adenovirus infection is important in surveillance and control of the infection. In this study, three monoclonal antibodies (MAbs) 5D4, 7B2 and 3D10 were generated against the recombinant HAdV-3 hexon protein. Western-blots analysis showed that all three MAbs recognized the native HAdV-3 hexon protein, but only 5D4 reacted with the native HAdV-3 virus particles by indirect enzyme-linked immunosorbent assay (ELISA). The epitopes recognized by these MAbs were further investigated by epitope analysis with a series of peptides of the hexon protein. The epitope for 3D10 was located to the sequence (339)GVLAGQASQLNA(350) and present in all the currently recognized serotypes of human adenovirus. The MAb 7B2 showed broad reactivity with the peptides from group B, group E, and most serotypes of group D, but not with those from group A, C and F adenoviruses. The epitope of 5D4, located in the hypervariable region 3 (HVR3), was only shared by HAdV-3 and HAdV-7. Further ELISA demonstrated that 5D4 only recognized HAdV-3 and HAdV-7 virus particles. The MAbs and the identified epitopes may play roles in clinical serotype-specific diagnosis, viral structure analysis, as well as in the identification of new serotypes of human adenovirus.