The interesterification of waste cooking oil (WCO) and ethyl acetate (ETA) are investigated in homogeneous catalyst system using sodium hydroxide (NaOH) and acetic acid (CH3COOH). Later on, the subsequent biofuel has been investigated for the combustion characteristics, gaseous and particulate matter related emissions in a single-cylinder agricultural diesel engine. In the reaction, fatty acid ethyl esters were the main product whereas triacetin by-product was formulated in a single phase biofuel. The parameters affected the free fatty acid (FFA) conversion were studied such as catalyst system, molar ratio of ETA:WCO, molar ratio of catalyst:WCO, reaction time, and temperature. The experimental results revealed that NaOH was more favorable than the acid catalyst counterpart. The 92% biofuel yield was reached from the optimization at the NaOH:WCO of 0.015:1 M ratio and the ETA:WCO of 30:1 M ratio at 80 degrees C in 3 h. In the engine validation at 3.4 and 6.6 bar IMEP loads, 1700 rpm speed, the results from the combustion analysis using an indicating system reveal that the WCO biofuel initiates the combustion faster with pronounce premixed combustion regime than that of diesel fuel. The specific fuel consumption of WCO biofuel was greater, leading to a slight reduction in brake thermal efficiency by 4% and 10% at 3.4 and 6.6 bar IMEP loads, respectively compared with diesel fuel. The nano-particle emissions was characterized by an electrical mobility spectrometer and analyzed in terms of particle number. The total particle number increased with smaller size when fueling with WCO biofuel. In comparison over the loads tested, the total particle number concentrations were in the ranges of 8.1 x 10(11) to 1.1 x 10(12) m(-3) for WCO biofuel and 4.8 x 10(11) to 5.7 x 10(11) m(-3) for diesel fuel. Meanwhile, the particle sizes were in the ranges of 182-251 nm for WCO biofuel and 279-402 nm for diesel fuel. Summarily, for the biofuel production, this homogeneous process is beneficial in terms of low-cost feedstock, glycerol-free and mild reaction condition.