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 CaF₂ substrate. FT-IR spectra obtained from ECM of tumor tissue exhibit stronger absorption bands in the spectral region from 1000 to 1200 cm⁻¹ and around 1750 cm⁻¹ 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.