Fast pyrolysis bio-oils (FPBOs) originating from forest residues contain extractive-derived substances, which may form a separate, sticky layer with char particles on the surface of these bio-oils. In this study, first, the removal of extractive-derived substances from the bio-oil top phase was studied by common solvents with different polarities. In this case, the results indicated that when aimed at the maximum yield of single-phase fuel oil and thus the maximum amount of extractives removed, the use of n-heptane or n-hexane seems to be of benefit for this purpose. For safety reasons, the use of n-heptane was recommended. Second, an analysis practice (extraction time and the way of mixing) was optimized. In order to reduce the extraction time and enhance the extraction yield, it was important to break the oil surface in extraction. Third, based on the characterization results of the n-heptane extract by gas chromatography and ultraviolet spectroscopy, the detected compounds were classified as fatty acids, resin acids, esterified fatty acids, terpenoids, and steroids, and their total content (27 wt %) was lower than that of lignin-derived compounds (70 wt %). The extraction of the FPBO top phase with n-heptane followed by this analysis practice was a useful way to estimate the content and composition of lipophilic extractives.