Bio-oils have been known with some advantages such as biodegradability, renewable, oxygen content, no-sulfur content. However, high viscosity, high surface tension and density due to the large structure may be the main causes strongly affecting the spray characteristics, mixture formation, combustion process, and emission characteristics of diesel engines running on bio-oils. In this work, the evaluation of the relationship between the spray parameters of used bio-oils including spray penetration (S) and cone angle (ɸ), and preheating temperature compared to fossil diesel fuel was conducted. Besides, the influence of the spray parameters on the breakup mechanism, brake thermal efficiency (ηe), heat release rate (HRR), and emission characteristics of an 80hp-diesel engine was reported. As a result, pure bio-oil was preheated to 105 °C to achieve the similarity of some physical properties and spray parameters compared to diesel fuel, but emission parameters of carbon monoxide (CO) and unburnt hydrocarbon (UHC) were 23.10% and 23.36% respectively higher. Meanwhile, brake thermal efficiency (ηe), and emissions of CO2, NOx, smoke were 3.36%, and 12.00%, 8.86%, 48.48% respectively lower than those of fossil diesel fuel. The findings in this paper showed further evidence in the direct use of pure biodiesel as a fuel for diesel engines. •The heating method is used to improve the disadvantages of bio-oils.•Spray characteristics of preheated bio-oils to 80–110 °C are determined.•Engine performance and exhaust gas temperature are measured and analyzed.•The optimum heating temperature for as-used bio-oils is 105 °C.•An exhaust gas-electricity integration system is fabricated for experimental purpose.