Abstract Emulsification characteristics of bio-oils and diesel at various operating conditions are analyzed. Three different commercial emulsifiers (i.e., Span 80, Tween 80, and Atlox 4914) and four bio-oils from fast pyrolysis of wood wastes are studied. When the three emulsifiers are individually employed, the performance of the emulsifiers is characterized by the order of Atlox 4914 > Span 80 > Tween 80. Atlox 4914, consisting of the hydrophilic portion of PEG and the hydrophobic portion of poly alkyd resin, has a better performance. The HLB value of an emulsifier can be obtained by blending Span 80 and Tween 80 or Span 80 and Atlox 4914. The optimum HLB for the emulsification of bio-oils and diesel linearly increases with increasing the atomic O/C or H/C ratio, water content, and decreasing higher heating value (HHV) of bio-oil. The correlation of the optimum HLB and HHV provides the best result, which can be employed for practical emulsification operation between bio-oils and diesel and is conduce to reducing operation cost. The functional groups in the emulsifiers, bio-oils, and diesel are analyzed by a Fourier transform infrared spectroscopy (FTIR) to recognize the characteristics of emulsions. It is found that FTIR is a fast and effective tool to examine the stability and homogeneity of emulsified fuels, even though no stratification is observed. Accordingly, this developed method can be used to rapidly and precisely identify the performance of emulsification in industry.