Sustainable energy from biomass is one of the most promising alternative energy sources and is expected to partially replace fossil fuels. Tobacco industries have normally rid their processing residues by landfilling or incineration, affecting the environment negatively. These residues can be used to either extract high-value chemicals or generate bio-energy via hydrothermal liquefaction. The main liquid product or bio-oil consists of highly complicated chemicals. In this work, the bio-oil from hydrothermal liquefaction of tobacco processing residues was generated in a batch reactor at biomass-to-deionized water ratio of 1:3, temperature of 310°C, and 15 min residence time, yielding the maximum liquid products for more than 50% w/w. The liquid products were analyzed, using two-dimensional gas chromatography and time-of-flight mass spectrometry (GC × GC/TOF MS). This technique allowed for a highly efficient detection of numerous compounds. From the results, it was found that hydrothermal liquefaction can cleave biopolymers (cellulose, hemicellulose, and lignin) in tobacco residues successfully. The hydrothermal liquefaction liquid products can be separated into heavy organic, light organic, and aqueous phase fractions. By GC × GC/TOF MS, the biopolymers disintegrated into low molecular weight compounds and classified by their chemical derivatives and functional groups could be detected. The major chemical derivative/functional groups found were cyclic ketones and phenols for heavy organic and light organic, and carboxylic acids and N-containing compounds for the aqueous phase. Additionally, by the major compounds found in this work, simple pathway reactions occurring in the hydrothermal liquefaction reaction were proposed, leading to a better understanding of the hydrothermal liquefaction process for tobacco residues.