During influenza infection, hemagglutinins on the viral surface bind to sialic acids on the host cell’s surface. Whilst all hemagglutinins bind sialic acids, human influenza targets terminal α2,6 sialic acids and avian influenza targets α2,3 sialic acids. For interspecies transmission (zoonosis), the hemagglutinin must mutate to adapt to these differences. Here, multivalent gold nanoparticles bearing either α2,6- or α2,3-sialyllactosamine have been developed to interrogate a panel of hemagglutinins from pathogenic human, low pathogenic avian and other species’ influenza. This method exploits the benefits of multivalent glycan presentation compared to monovalent presentation, to increase affinity, and investigate how the multivalency affects selectivity. Using a library-orientated approach, parameters including polymer coating and core diameter were optimised for maximal binding and specificity were probed using galactosylated particles and a panel of biophysical techniques (UV-Vis, dynamic light scattering and biolayer interferometry). The optimized particles were then functionalized with sialyllactosamine and their binding analysed against a panel of hemagglutinins derived from pathogenic influenza strains including low pathogenic avian strains. This showed significant specificity crossover, which is not observed in monovalent formats, with binding of avian hemagglutinins to human sialic acids and vice versa in agreement with alternate assay formats. These results demonstrate that precise multivalent presentation is essential to dissect the interactions of hemagglutinins and may aid the discovery of new tools for disease and zoonosis transmission.