This chapter introduces the physical properties of bio-oil and the corresponding characterization methods. The properties of bio-oil include density, water content, pH value, viscosity, gross calorific value, ultimate analysis, ash and solid content, thermal sensibility, combustion characteristics, etc. Bio-oil composition can be characterized by GC-MS, HPLC, FTIR and NMR, etc. In addition, adopting proper pretreatment methods such as fractional condensation, membrane separation, centrifugal separation, extraction, column chromatography and distillation can realize partial enrichment of special compounds in some fractions, which will benefit the characterization of bio-oil. Some compounds, such as phenolic compounds, sugars and aldehydes, have unique characteristics, and their quantitative analysis could be achieved by previous separation and derivatization of bio-oil. With the aid of a precise description of bio-oil properties and characterization, graded upgrading of bio-oil could be efficiently developed to obtain a high quality liquid fuel.