Human cytomegalovirus (HCMV) is a kind of herpesvirus which is widely infected all over the world. It is also the most common pathogen that causes fetal congenital malformations and death of organ transplant recipients. Some studies showed that gH protein was the main neutralizing antigen of HCMV, and specific anti-gH antibodys had the potential to neutralize HCMV and block virus transmission between cells. In this study, the gH gene fragment without signal peptide and transmembrane region was amplified, whose template was the BAC containing the whole genome of human cytomegalovirus clinical strain Toledo, and inserted into the expression vector to construct the recombinant expression plasmid pET32a′-gH. The recombinant plasmid with correct sequence identified was transformed into the expression strain TransB, and then TransB was induced to express the recombinant protein. Eight-week-old male BALB/c mice were immunized with purified recombinant gH protein. Five cell lines stably secreting anti-gH monoclonal antibodies were obtained by hybridoma technique, which were named 8A9, 8B4, 8C4, 8D9 and 8D12 respectively. All the five monoclonal antibodies had good reactivity to gH protein. Among them, 8A9, 8B4, 8D9 and 8D12 had certain virus capture ability, and the capture ability of 8D9 and 8D12 was stronger. In this study, we obtained anti-gH monoclonal antibodies with independent intellectual property rights, and the antibodies had good virus capture ability. On this basis, we will further identify whether they are neutralizing antibodys, which will lay a foundation for the development of neutralizing antibodies for the treatment of HCMV infection in the future.