A monoclonal antibody, 3BIgG, against the prokaryotically expressed foot-and-mouth disease virus (FMDV) non-structural protein (NSP) 3B was obtained. The 3BIgG-sepharose conjugant (3BmAb-6BFF) was prepared by adding the purified 3BIgG into epoxy-activated sepharose 6BFF, incubating with the inactivated FMDV, and then removing the sepharose by centrifugation. The vaccine was made from the supernatant emulsified with oil-adjuvant ISA206. Ten guinea pigs, 26 pigs and six cattle were vaccinated, and a vaccination control group was included without treatment with 3BmAb-6BFF. After 28 days, 9/10 pigs challenged with FMDV were protected, this result was the same as the control group, indicating that the vaccine potency was not reduced after treatment with 3BmAb-6BFF. The other animals were vaccinated weekly for nine weeks, and serum samples were collected to detect 3ABC-antibody titers. The results showed that 3ABC-antibody production was delayed and the positive antibody rates were lower when vaccination was carried out using vaccines treated with 3BmAb-6BFF compared with untreated vaccines. The findings of this study suggest that it is possible to reduce NSPs using a mAb-sepharose conjugant in FMD vaccines without reducing their efficacy.