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Abstract Oesophageal cancer (OC) is associated with high morbidity and mortality, and surgery is the most effective approach to treat it. In order to reduce surgical risks and duration of surgery, we explored a new strategy to determine tumour margins in surgery. In this study, we included 128 cancerous and 128 noncancerous database entries obtained from 32 human patients. Using internal extractive electrospray ionization-MS, in positive ion detection mode, the relative abundances of m/z 104.13, m/z 116.10, m/z 132.13, and m/z 175.13 were higher in cancer tissue while the relative abundances of m/z 82.99, m/z 133.11, m/z 147.08, m/z 154.06, and m/z 188.05 were higher in normal tissue. Using partial least squares analysis, the mass spectra of cancer samples was discriminated from those of normal tissues, and the discriminatory ions were obtained from loading plots. Dimethylglycine(m/z 104), proline(m/z 116), isoleucine(m/z 132), asparagine(m/z 133), glutamine(m/z 147), and arginine(m/z 175) were identified by collision-induced dissociation experiments. Using the ROC curve analysis, we verified the validity of six amino acids for the identification of tumour tissue. Further investigations of tissue amino acids may allow us to better understand the underlying mechanisms involved in OC and develop novel means to identify tumour tissue during operation.