Three difunctional ethyl esters (DFEEs), including ethyl 16-hydroxyhexadecanoate, ethyl (Z)-18-hydroxyoctadec-9-enoate, and diethyl (Z)-octadec-9-ene-1,18-dioate, were isolated as nearly pure compounds from sawdust-derived bio-oil. Their structures were tentatively identified by gas chromatography/mass spectrometry (GC/MS) and high-resolution mass spectrometry (HR-MS). Combination transesterification reaction of DFEEs to difunctional methyl esters (DFMEs) and determination of the GC/MS retention time differences of DFEEs and corresponding DFMEs is a simple method to determine the ester group number of unknown DFEEs. GC/MS results indicate that the fragment ions at mass-to-charge ratios (m/z) of 88 and 98 are typical ions for these DFEEs. The former ion is formed via well-known McLafferty rearrangement, whereas the formation pathway of the ion at m/z 98 is unreported before. HR-MS results show that the fragment ion at m/z 98 should be a cyclohexanone radical ion, which is possibly formed via a novel rearrangement initiated by remote hydrogen rearrangement. Besides long-chain DFEEs, long-chain difunctional carboxylic acids (including dicarboxylic acids, omega-hydroxy acids, diacid monomethyl ester, and diacid monoethyl ester) and DFMEs (including diacid dimethyl ester and omega-hydroxy methyl ester) are also typical of the fragment ion at m/z 98. Hence, it is a common characteristic to form the rearrangement ion at m/z 98 for long-chain difunctional carboxylic acids, DFEEs, and DFMEs, and this special mass spectral rearrangement could facilitate the identification of these classes of compounds. Using this rearrangement rule of DFEEs, three omega-hydroxy ethyl esters, including ethyl 22-hydroxydocosanoate, ethyl 24-hydroxytetracosanoate, and ethyl 26-hydroxyhexacosanoate, were tentatively identified by GC/MS.