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In this work, we developed a rapid and high-sensitivity method for simultaneous analyses of fatty alcohols, fatty aldehydes, and sterols by combining the optimized derivatization reaction with electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS). Pyridine and thionyl chloride were used as derivatization reagents as they were easily removed after the derivatization reaction and could generate permanently charged tags on different functional groups including hydroxyls and aldehydes. Through this one-step derivatization reaction, the sensitivity of detection for fatty alcohols, fatty aldehydes, and sterols was significantly increased. Moreover, the introduction of ion mobility spectrometry (IMS), offering an additional resolution power, ensured more sensitive and accurate detection of derivative products without increasing analytical time. Being connected with high-performance liquid chromatography, more than 15 kinds of compounds were analyzed within 4 min. Relative quantification using peak intensity ratios between d 0-/d 5-labeled ions were subsequently applied for analyzing these 15 kinds of compounds in human thyroid carcinoma and para-carcinoma tissues. The results showed significant differences in content of some analytes between these two kinds of tissues (p < 0.05). The correlations between most of the analytes in thyroid carcinoma tissues are better than the correlations in para-carcinoma tissues.