To explore the feasibility of quick intraoperative in situ and noninvasive diagnosis of lymph node metastasis in gastric cancer by Fourier transform infrared (FTIR) spectrometry. FTIR spectra of surgically removed fresh lymph nodes were measured by FTIR via probe of attenuated total reflection (ATR). For each spectrum, 13 bands were indentified and assigned between 3 000 and 1 000 cm-1. Peaks in the spectra were measured and relative intensity ratios were calculated and compared between the spectra of Metastatic lymph nodes (MLN) and Non-metastatic lymph nodes(NMLN). Standard statistic analysis was performed. 720 lymph nodes were measured in 38 gastric cancer patients. Results show that there were significant differences between the FTIR of 540 MLN and 180 NMLN. (1) For the band related to nucleic acid: The ratios of I1 240/I1 460(p=0.015) and I1 080/I1 460(p=0.034)increased in MLN, which shows that the relative quantity of nucleic acid was more in MLN than that in NMLN. (2) For the bands related to protein: The ratios of I1 640/I1 460(p=0.001) and I1 546/I1 460(p=0.027) increased in MLN, which shows that the relative quantity of protein was more in MLN. (3) For the bands related to lipid: The ratio of I2 855/I1 460 and I1 740/I1 460 decreased in MLN FTIR spectrum, indicating the lower relative quantity of lipid in MLN. (4) For the bands related to carbohydrate: The ratio of I1 160/I1 460(p=0.023) decreased in MLN FTIR spectrum, indicating the lower relative quantity of carbohydrate in MLN. The results demonstrate that the FTIR spectroscopy technique maybe develop into a promising method for in situ and quick intraoperative differential diagnosis of lymph node metastasis in gastric cancer.