Fourier transform infrared (FT-IR) spectroscopy was applied to characterize the extracellular matrix (ECM) of kidney tumor tissue and normal kidney tissue. Freshly resected tissue samples from 31 patients were pressed on a substrate. FT-IR spectra obtained from ECM of tumor tissue exhibit stronger absorption bands in the spectral region from 1000 to and around than those obtained from normal tissue. It is likely that the spectra of ECM of kidney tumor tissue with large increases in the intensities of these bands represent a higher concentration of fatty acids and glycerol. Amide I and amide II bands are stronger in the spectra of ECM from normal tissue, indicating a higher level of proteins. Our results suggest that FT-IR spectroscopy of the ECM is an innovative emerging technology for real-time intraoperative tumor diagnosis, which may improve margin clearance in renal cancer surgery.