To better understand the antibody landscape changes following influenza virus natural infection and vaccination, we developed a high-throughput multiplex influenza antibody detection assay (MIADA) containing 42 recombinant hemagglutinins (rHAs) (ectodomain and/or globular head domain) from pre-2009 A(H1N1), A(H1N1)pdm09, A(H2N2), A(H3N2), A(H5N1), A(H7N7), A(H7N9), A(H7N2), A(H9N2), A(H13N9), and influenza B viruses. Panels of ferret antisera, 227 paired human sera from vaccinees (children and adults) in 5 influenza seasons (2010 to 2018), and 17 paired human sera collected from real-time reverse transcription-PCR (rRT-PCR)-confirmed influenza A(H1N1)pdm09, influenza A(H3N2), or influenza B virus-infected adults were analyzed by the MIADA. Ferret antisera demonstrated clear strain-specific antibody responses to exposed subtype HA. Adults (19 to 49 years old) had broader antibody landscapes than young children (<3 years old) and older children (9 to 17 years old) both at baseline and post-vaccination. Influenza vaccination and infection induced the strongest antibody responses specific to HA(s) of exposed strain/subtype viruses and closely related strains; they also induced cross-reactive antibodies to an unexposed influenza virus subtype(s), including novel viruses. Subsequent serum adsorption confirmed that the cross-reactive antibodies against novel subtype HAs were mainly induced by exposures to A(H1N1)/A(H3N2) influenza A viruses. In contrast, adults infected by influenza B viruses mounted antibody responses mostly specific to two influenza B virus lineage HAs. Median fluorescence intensities (MFIs) and seroconversion in MIADA had good correlations with the titers and seroconversion measured by hemagglutination inhibition and microneutralization assays. Our study demonstrated that antibody landscape analysis by the MIADA can be used for influenza vaccine evaluations and characterization of influenza virus infections.| Repeated influenza vaccination and natural infections generate complex immune profiles in humans that require antibody landscape analysis to assess immunity and evaluate vaccines. However, antibody landscape analyses are difficult to perform using traditional assays. Here, we developed a high-throughput, serum-sparing, multiplex influenza antibody detection assay (MIADA) and analyzed the antibody landscapes following influenza vaccination and infection. We showed that adults had broader antibody landscapes than children. Influenza vaccination and infection not only induced the strongest antibody responses to the hemagglutinins of the viruses of exposure, but also induced cross-reactive antibodies to novel influenza viruses that can be removed by serum adsorption. There is a good correlation between the median fluorescence intensity (MFI) measured by MIADA and hemagglutination inhibition/microneutralization titers. Antibody landscape analysis by the MIADA can be used in influenza vaccine evaluations, including the development of universal influenza vaccines and the characterization of influenza virus infections.