Influenza A virus (IAV) remains a major threat to global health. The emergence of IAV strains resistant to commonly used anti-influenza medicines has made the need for novel broad-spectrum antiviral agents more urgent. The IAV nucleoprotein (NP) is one of the most conserved viral proteins encoded by IAV and is essential for multiple processes in the lifecycle of IAVs. In this study, we screened a high-affinity nanobody, Nb7, which specifically recognizes NP from different subtypes of IAV and broadly inhibits the replication of various IAVs. Mechanistically, Nb7 blocked the nuclear export of viral ribonucleoprotein (vRNP) complexes and the subsequent assembly of progeny virions by specifically binding the nuclear export sequence 1 (NES1) region of the NP. The conserved Q42/E46/K48 residues within the NES1 region of NP were identified as the key sites for the binding of Nb7 and were synergistically responsible for the nuclear export of vRNP. In addition, Nb7 fused with a trans-activating transduction peptide efficiently inhibited the replication of IAV in vitro and provided full protection against lethal IAV infection in a mouse model. Taken together, our findings revealed that the NP-specific Nb7 is a promising candidate against IAV infection. This study provides a new understanding of the nuclear export of IAV-vRNP and contributes to the development of broad-spectrum anti-influenza approaches.