Rapid identification of pathogens is the vital step for prevention and control of respiratory viruses. Application of novel nanobodies holds significant promise for highly sensitive pathogen detection methods, but they face structural instability in applications. Here, we prepared the multivalent nanobodies with improved affinities for antigen and developed a multivalent nanobodies-based Surface-enhanced resonance Raman scattering-lateral flow immunochromatographic assay (SERS-LFA) for highly sensitive detection of influenza A viruses (IAVs). Notably, two dimensional MoS2@Au nanosheets were taken as the substrate of immune labels, with large specific surface area and superior SERS performance. Furthermore, 2D MoS2@Au nanosheets conjugated large number of multivalent nanobodies based on the biotin-streptavidin amplification system. The sensitivity of established SERS-LFA for the recombinant nucleoproteins of IAVs and inactive IAVs were as low as 1.7 pg/mL and 0.15 pfu/mL, respectively, with excellent specificity and reproducibility. Furthermore, 52 clinical throat samples were tested by our proposed SERS-LFA, 100% consistent with RT-PCR, exhibiting higher reliability than commercial gold-based LFA.