To explore the feasibility of quick intraoperative in situ and noninvasive diagnosis of cholangiocarcinoma by Fourier transform infrared(FTIR)spectroscopy, 26samples of freshly resected cholangiocarcinoma tissues and 43 samples of benign bile duct tissue were collected during surgery. And all fresh samples were measured by FTIR spectroscopy via probe of attenuated total reflection(ATR)without pretreatment immediately after resection. For each spectrum, 12 bands were identified and assigned between 3800 and 1000cm^-1. The peak position was found, and the intensity and area of the bands were measured; The ratios of the intensity and area were calculated. Standard statistic analysis was performed. The results illustrated that the FTIR spectra of malignant tissue were significantly different from those of benign tissue: 1. The bands related to lipid: The peak of 2925cm^-1 shifted to lower wave number significantly(P=0.033); 2. The bands related to nucleic acid and lipids:the ratios of I₁₀₈₃/I₁₄₆₀(P=0.005), S₁₀₈₃/S₁₄₆₀(P=0.001)and S₁₂₄₀/S₁₄₆₀(P=0.025)raised significantly, indicating that the relative quantity of nucleic acid to lipids had evidently increased in malignancy. 3. The bands related to protein: The ratios of I₁₅₅₀/I₁₀₈₃(P=0.000)and S₁₅₅₀/S₁₀₈₃(P=0.000)reduced significantly, implying that the relative quantity of protein to nucleic acid in malignancy decreased significantly and the secondary structure of proteinchanged probably. Our primary result illustrated that the FTIR spectroscopy technique maybe a promising method for in situ and quick intraoperative diagnosis of cholangiocarcinoma and has great value for clinical application.