Ammonia is a promising alternative fuel that can replace current fossil fuels. Hydrogen carrier, zero carbon base emissions, liquid unlike hydrogen, and can be produced using renewable resources, making ammonia a future green fuel for the internal combustion engine. This study aims to show the procedure of utilizing ammonia as a primary fuel with biodiesel in a dual-fuel mode. Hence, a single-cylinder diesel engine was retrofitted to inject ammonia into the intake manifold, and then a pilot dose of biodiesel is sprayed into the cylinder to initiate combustion of the premixed ammonia-air mixture. The effects of various ammonia mass flow rates with a constant biodiesel dose on engine performance and emissions were investigated. Furthermore, a one-dimensional model has been developed to analyze the combustion of ammonia and biodiesel. The results reveal that 69.4% of the biodiesel input energy can be replaced by ammonia but increasing the ammonia mass flow rate slightly decreases the brake thermal efficiency. Moreover, increasing the ammonia load contribution significantly reduced the emissions of CO2, CO, and HC but increased the emission of NO. It was found that ammonia delayed the start of combustion by 2.6CAD compared with pure biodiesel due to the low in-cylinder temperature and the high resistance of ammonia to autoignition. However, the combustion duration of biodiesel/ammonia decreased 19CAD compared with only biodiesel operation at full load, since most of the heat was released during the premixed combustion phase.