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Vibrational spectroscopy provides the possibility for sensitive and precise detection of chemical changes in biomolecules due to development of cancers. In this work, label-free near-infrared surface enhanced Raman spectroscopy (SERS) was applied for the differentiation between cancerous and normal human bladder tissues via analysis of the extracellular fluid of the tissue. Specific cancer-related SERS marker bands were identified by using a 1064 nm excitation wavelength. The prominent spectral marker band was found to be located near 1052 cm –1 and was assigned to the C–C, C–O, and C–N stretching vibrations of lactic acid and/or cysteine molecules. The correct identification of 80% of samples is achieved with even limited data set and could be further improved. The further development of such a detection method could be implemented in clinical practice for the aid of surgeons in determining of boundaries of malignant tumors during the surgery.