Foot-and-mouth disease (FMD) is an endemic, highly contagious, and devastating disease of livestock production in Ethiopia. Control of this disease relies mainly on prophylactic vaccination by willing farmers without a countrywide vaccination program. The objectives of this study were to quantify the humoral immune response and evaluation of the serological relationship of the vaccine strain used with representative field strain isolates. This was performed by primo vaccination of 6-9-month-old Holstein Friesian calves (35 treatment and 4 control calves) on day one and booster vaccination on day 28. Calves were vaccinated using the locally available National Veterinary Institute (NVI), Bishoftu, Ethiopia, inactivated aluminum hydroxide adjuvant monovalent (either O, A, SAT-2 alone) or trivalent (combination of A, O, SAT-2) vaccine (A/ETH/6/2000 (G-VII, O/ETH/38/2005(EA-3) and SAT-2/ETH/64/2009(XIII)). A 2 mL or 4 mL dose was used to vaccinate all calves except the animals that served as a control. In the case of the trivalent vaccine, a 4 mL dose was used to vaccinate calves. The serum was collected at 7, 14, 21, 28, and 56 days post-vaccination (d.p.v.). The humoral immune response was quantified by the solid-phase competitive enzyme-linked immunosorbent assay (SPC ELISA) and the virus-neutralization test (VNT). The serological relationship of heterologous and homologous viruses was also evaluated by adjuvant vaccine matching tests. The r1-value was determined using serum collected 21 d.p.v. An increase in immune response was observed from 7 d.p.v. to 28 d.p.v. in calves who received a 4 mL dose containing a 107.24 antigen load of 100 tissue culture infective dose (100TCID50) virus titer in the formulation. Upon receiving a booster dose on day 28, the humoral immune response was checked on the 56th day post-initial vaccination. Amounts of 54%, 72%, 79%, and 72% of inhibition for A, O, SAT-2, and trivalent vaccine in the three serotypes SPCE, respectively, was measured. Here, it was found that the immune response of calves increased from day 7 to 56, as evidenced by SPCE analysis. Likewise, an increase in antibody titer measured by a one-dimensional virus neutralization assay was also in line with SPCE analysis. This indicates that the vaccine is capable of inducing a neutralizing antibody that confers a protective immune response in 70%, 62%, and 100% heterologous field strains of A, O, and SAT-2 isolates, respectively, and has an average antigenic relationship of >0.3 with a standard deviation of +0.05 (N = 3) to the vaccine strains A/ETH/6/2000, O/ETH/38/2005 and SAT-2/ETH/64/2009, respectively, when using the one-dimensional virus neutralization test. The contribution and importance of this study is a confirmation of the vaccine and the field strain serological relationship for serotype SAT-2 strain and further research/change of vaccination strategy/ improvement in the currently used vaccine to cover a wide range of prevailing genotypes/lineages and induction of sound immune response after vaccination for serotype A and O strain. This study suggests that the trivalent vaccine produced by the National Veterinary Institute containing viral isolates from serotype O, A, and SAT-2 has a good serological relationship with the majority of circulating field strains in Ethiopia.