The authors systemically reviewed the fast development of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and its clinical application in the past decades. The advantages of this objective technique include real time scanning, easy manipulation and no harm to the subjects examined. Combined with pattern recognition methodology and further confirmation with the clinical and pathological diagnosis, the goal of fast differentiation of malignancy from benign lesions could be achieved. ATR-FTIR spectroscopy technique has shown high differential capacity for benign and malignant tissues such as thyroid, breast and pulmonary diseases. ATR-FTIR spectroscopy has being applied in investigating the differential value (the sensitivity, specificity, and accuracy) of metastatic lymph nodes in thyroid and breast cancer with encouraging results. ATR-FTIR technique would become a promising tool in tissue diagnosis intra-operatively. ATR-FTIR spectroscopy has also been widely applied in detecting bio-fluid to differentiate diseases. The serum ATR-FTIR spectroscopy has the ability of reflecting disease-related information in a fingerprint manner with little amount of blood. Several published articles have covered diseases such as glioma, chest pain, prostate cancer, renal failure, Alzheimer’s disease, and ovarian cancer. The results of these researches have proved the efficacious discriminate value of this method. As ATR-FTIR spectroscopy has the potential of fast analysis, accurate diagnosis, and low cost-effective value. It would become one of the most important assisting diagnosis tools in future. Follow-up study should focus on enhancing sample quality and enlarging sample size to have further prospective clinical application.