Detection of human adenoviruses (HAdVs) in nasopharyngeal swab samples by immunofluorescence assay (IFA) will be valuable for diagnosing HAdV infection， which is a leading cause of severe respiratory tract disease， and will help in curbing the spread of HAdV. Monoclonal antibodies employed in IFA for HAdV detection should ideally target highly conserved epitope types. Here， we describe the development of two antigen-binding fragments (Fabs) with specific reactivity to HAdV using phage antibody library technology. When tested with IFA， both Fabs recognized cells infected with several types of HAdV， some of which have been identified in epidemics globally， or associated with outbreaks of severe or fatal acute respiratory diseases. The specificity and cross-reactivity of both Fabs to HAdVs indicated that the generated Fabs could be applied in the development of IFAs to detect HAdVs. Both Fabs bound to the knob proteins， as shown by chemiluminescence enzyme immunoassay and western blot. In addition， epitope mapping showed that both Fabs recognized a conserved linear epitope among several types of HAdV. Two different Fabs recognized the same epitope， suggesting that the epitope triggered the production of at least two kinds of antibodies in the body. The generated Fabs exerted no neutralization against HAdVs. The results demonstrate that both Fabs bind to an epitope that plays no role in neutralization of HAdV.