Abstract Background Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus (sensu stricto), is a life-threatening but neglected zoonosis. Glycolytic enzymes are crucial molecules for the survival and development of E. granulosus. The aim of this study was to investigate the molecular characterization, immunogenicity, tissue distribution and serodiagnostic potential of E. granulosus hexokinase (EgHK), the first key enzyme in the glycolytic pathway. Methods EgHK was cloned and expressed in Escherichia coli. Specific serum antibodies were evaluated in mice immunized with recombinant EgHK (rEgHK). The location of EgHK in the larval stage of E. granulosus was determined using fluorescence immunohistochemistry, and the potential of rEgHK as a diagnostic antigen was investigated in patients with CE using indirect enzyme-linked immunosorbent assay (ELISA). Results Recombinant EgHK could be identified in the sera of patients with CE and in mouse anti-rEgHK sera. High titers of specific immunoglobulin G were induced in mice after immunization with rEgHK. EgHK was mainly located in the tegument, suckers and hooklets of protoscoleces and in the germinal layer and laminated layer of the cyst wall. The sensitivity and specificity of the rEgHK-ELISA reached 91.3% (42/46) and 87.8% (43/49), respectively. Conclusions We have characterized the sequence, structure and location of EgHK and investigated the immunoreactivity, immunogenicity and serodiagnostic potential of rEgHK. Our results suggest that EgHK may be a promising candidate for the development of vaccines against E. granulosus and an effective antigen for the diagnosis of human CE. Graphical Abstract