The detection and evaluation of concentration of influenza virus proteins in biological samples is critical in a broad range of medical and biological investigations regarding the concern over potential outbreaks of virulent influenza strains in animals and humans. This paper describes a sensitive, label-free approach for the detection of a virulence factor PB1-F2. PB1-F2 is a small, 90 amino acid long polypeptide expressed in influenza A viruses, which generally exacerbate virus pathogenicity. The developed immunosensoris based on a non-the-chipcovalently immobilized specific monoclonal anti-PB1-F2 antibody and a SPR technology. The immunosensor was calibrated using purified full length PB1-F2 protein. Itdetected PB1-F2 with the linear range extended from 10 to 500 nM, repeatability of 5% for 500 nM PB1-F2 and showed saturationof protein concentrations higher than 1 μM. The sensor can quantify PB1-F2 in its monomeric form but not when its oligomerization was induced by preincubation in 0.05% SDS. The immunosensor was successfully applied in the detection and quantification of PB1-F2 in infected mouse lungs and cell lines, providing temporal expression profiles of PB1-F2 during viral infection. In lungs of infected mice, the influenza virus structural nucleoprotein NP was detected in parallel using a specific anti-NP antibody. This parallel detection of PB1-F2 and NP suggests that applied sensor chip technology may be amenable to an arrow immunosensor for simultaneous detection of all known influenza virus proteins in infected tissues and cells.