Dengue infection is one of the major health problems speading worldwide including in Indonesia. The dengue hemorrhagic fever (DHF) incidence in Indonesia has increased rapidly from 1968 until 2013. However, the case fatality ratio decreased during the same period due to early detection of dengue infection by using monoclonal antibody and non-structural 1 (NS1) protein biomarker. This study was conducted to clone the heavy chain of fragment antigen binding (Fab) using local hybridoma clone 71E2 secreting monoclonal antibody anti-NS1, which was induced by the dengue virus. Polymerase chain reaction was used to amplify the heavy chain gene of Fab and agarose gel electrophoresis was used for visualization. The recombinant plasmid that consists of the heavy chain gene and pTA2 vector was transformed to Escherichia coli TOP10. Polymerase chain reaction, digestion, and agarose gel electrophoresis was then used to confirm the gene insertion in the recombinant plasmid. A band of 550 bp appeared in the PCR product of recombinant plasmid, indicating the heavy chain gene of Fab from clone 71E2 has been inserted into the pTA2 vector successfully.