The aim of the research is to estimate Fourier transform infrared spectroscopy (FTIR) as a novel diagnostic tool for rectal cancer metastatic lymph nodes. 160 freshly normal and malignant lymph nodes were collected from 80 rectal cancer patients for spectrum analysis. FTIR spectra were generated from tissues samples using infrared wavenumber from 4 000 to 1 000 cm-1 region. The ratios of spectral intensity and relative intensity ratios (I/1 460) were calculated. Principal component analysis (PCA) and Fisher’s Linear discriminant analysis (LDA) were applied to distinguish the malignant from normal. From 4 000 to 1 700 cm-1, principal components 1 (PC1) was 3 260 cm-1 and PC2 was 1 740 cm-1; from 1 700 to 1 000 cm-1, PC1 was 1 640 cm-1 ab PC2 was 1 080 cm-1. Four prominent significant wavenumbers at 1 080 , 1 640, 1 740 and 3 260 cm-1 separated cancer spectra from normal spectra into two distinct groups using PCA. T test associated with relative intensity ratios (I1 080/I1 460, I1 640/I1 460, I3 260/I1 460, I1 740/I1 460) revealed that these wavenumbers were also able to distinguish cancer and normal lymph nodes spectra. The bands 3 260, 1 640, 1 550, 1 080 and 1 740 cm-1, associated with Protein, nucleic acid and lipid. Compared with normal lymph nodes tissues, malignat lymph nodes significant increased (p<0.05), but 1 740 cm-1 decreased. Six parameters (wavenumbers 1 080 and P1 300 cm-1, relative intensity ratios I1 080/I1 460, I1 640/I1 460, I3 260/I1 460, I1 740/I1 460) were selected as independent factors to perform discriminant functions. The sensitivity for PCA/LDA mode in diagnosing lymph nodes was 87.5% by discriminant analysis. The results demonstrate that FTIR is as a useful technique for detection malignant and normal lymph nodes, FTIR may be applied in clinical as a noninvasive, high sensitivity and specificity method for lymph nodes diagnosis.