The core focus of the present investigation is regarding biodiesel production from industrial hemp seed oil applying single-stage homogenous catalyzed transesterification process obtaining high yield of methyl ester. The engine tests were carried out on a single-cylinder, four-stroke, water-cooled, unmodified diesel engine operating with hemp seed oil methyl ester as well as its blends with conventional diesel fuel. The experimental findings of the test fuels were compared with those from diesel. The results pointed out that the performance and combustion behaviors of biodiesel fuels are just about in line with those of diesel fuel propensity. The specific fuel consumption for 5% biodiesel blend (0.291 kg/kW h), 10% biodiesel blend (0.305 kg/kW h), and 20% biodiesel blend (0.312 kg/kW h) blends at full load was closer to diesel (0.275 kg/kW h). In the meantime, the thermal efficiency for biodiesel was found to be at the range of 15.98-24.97% and it was slightly lower than that of diesel (18.10-29.85%) at the working loads. On the other hand, the harmful pollutant characteristics of carbon monoxide, hydrocarbon, and smoke opacity for biodiesel and its blends were observed to be lower in comparison with diesel during the trials. However, the oxides of nitrogen emissions for biodiesel were monitored to be as 6.85-15.40 g/kW h which was remarkably higher than that of diesel (4.71-8.63 g/kW h). Besides that, the combustion behaviors of biodiesel and its blends with diesel showed much the same followed those of diesel. Namely, the duration of ignition delay of biodiesel-diesel blends was shorter than that of diesel fuel because of the higher cetane number specification of the methyl ester. The highest gas pressures inside the cylinder as well as the rates of the heat release of biodiesel including test fuels are lower in contrast to the diesel due to the shorter ignition delay. It could be concluded that the utilization of biodiesel produced from industrial hemp seed oil in the diesel engine up to 20% (by vol.) will decrease the consumption of diesel and environmental pollution, especially in developing countries.