The study was conducted in Oromia Regional State of East Shoa Zone, Bishoftu town from October 2019 to June 2020 and to evaluate immune response of polyvalent FMD vaccine. Thirty-four cattle were used for longitudinal study and grouped into two ((29) vaccinated and (5) unvaccinated) groups. Twenty-nine cattle were vaccinated with locally available commercial inactivated polyvalent Foot and mouth disease (FMD) virus vaccine that was produced in National Veterinary Institute. Sera were obtained on 0, 14, 21, 35, 80 and 125 days post-vaccination and they were tested by Virus Neutralization Test and 3ABC ELISA. In all vaccinated cattle a significant increase in neutralizing antibody against structural protein was observed on day 14 post-vaccination for all the three seed strain virus (A/ETH/21/2000, p = 0.015; O/ETH/38/2005, p = 0.017; SAT2/ETH/64/2009, p = 0.007). On day, fourteen of post-vaccination vaccinated group showed immune response equal or above 1.5 log10 in a proportion of 69%, 73% and 94% for serotype A, O and SAT2 respectively. The optimum antibody titer was recorded on 35 day post-vaccination. The status of raised antibody titer on day 125 post-vaccination showed decreasing by 14% for serotype A, 18% for serotype O and 4% for serotype SAT2. The 3ABC ELISA for Differentiating infected from vaccinated animal test indicated that immune response to Non-structural protein was undetected in the vaccinated group of cattle. In unvaccinated group antibody titer was unrecorded to both structural protein and non-structural protein. In conclusion, commercially available FMD vaccine containing serotype A, O and SAT2 after first shoot induces immune response to structural protein rather than non-structural protein. This result suggests that FMD vaccines produced at National Veterinary Institute are Compatibility with DIVA test. However, further work required to enhance the strength and duration of immune response of the vaccine. Keywords; cattle, Foot and Mouth Disease, immune response and vaccine.