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Junin virus (JUNV) is the pathogen of Argentine haemorrhagic fever which results in high lethality, while there is limited therapeutics available. Here, a series of mouse monoclonal antibodies (mAbs) were isolated through immunization with DNA and screening against glycoprotein complex (GPC). Finally, five mAbs were found to effectively neutralize JUNV. Further research indicated that they were capable of binding conformational GPC and strongly binding glycoprotein 1 which is responsible for receptor recognition. Epitope mapping revealed that they targeted loop 3 of GP1, and Tyr122 and Asp123 in loop 3 were identified as their common binding sites, which may account for their neutralizing activity. This study presents a new strategy for developing neutralizing antibodies against JUNV and provides therapeutic candidates for protection against JUNV infection.Monoclonal antibodies were obtained based on DNA immunization and intact GPC screening.Five monoclonal antibodies effectively neutralize Junin virus.The neutralizing antibodies blocks Tyr122 and Asp123 in loop 3 of GP1.