Cattle were not previously thought susceptible to highly pathogenic avian influenza (HPAI) H5N1 virus infection until March 2024, when outbreaks were reported among dairy cattle. Data are sparse regarding the immune response of cattle to infections with such viruses. To better understand the systemic humoral immune responses of HPAI H5N1-infected dairy cattle, we conducted a prospective study of 15 cows on an H5N1-affected farm. The cows’ sera were studied using microneutralization (MN), hemagglutination inhibition, and enzyme-linked immunosorbent assays. At four months post-outbreak, MN assays revealed 73% (11/15) of the cattle sera had elevated H5 antibody levels that ranged from 1:40 to 1:640. Eight months post-outbreak, 73% retained elevated MN antibodies, wherein 18% (2/11) had an increased response, 63.6% (7/11) maintained antibody levels, and 18% (2/11) showed a decline. By 13 months post-outbreak, only 50% (5/10) retained elevated titers by the MN assay. The results from all three assays were in agreement; however, the MN assay seemed to be more sensitive. Our findings indicate a correlation among the three assays, with high levels of H5 antibodies persisting for more than a year after natural infections. This information is vital for guiding future use of H5 vaccines in dairy cattle farms.