Bio‐oils, produced by biomass pyrolysis, have become promising candidates for feedstocks of high value‐added chemicals and alternative sources for transportation fuels. Bio‐oil is such a complicated mixture that contains nonpolar hydrocarbons and polar components which cover almost all kinds of organic oxygenated compounds such as carboxylic acids, alcohols, aldehydes, ketones, esters, furfurals, phenolic compounds, sugar‐like material, and lignin‐derived compounds. Comprehensive characterization of bio‐oil and its subfractions could provide insight into the conversion process of biomass processing, as well as its further utilization as transportation fuels or chemical raw materials. This review focuses on advanced analytical strategies on in‐depth characterization of bio‐oil, which is concerned with gas chromatography, high‐resolution mass spectrometry, FTIR spectroscopy and NMR spectroscopy, offering complementary information for previous reviews.