Tumor necrosis factor-alpha (TNF-alpha), also known as cachectin, is one of the most important regulatory cytokines and mediates a variety of cell functions, including the stimulation of nitric oxide (NO) production which has been related to oxidative stress and diseases such as arthritis, diabetes, stroke, and chronic inflammation. Determination of TNF-alpha concentration in human serum might be helpful in the staging and prognosis of diseases. And it is also very important for the understanding of tumor biological processes, inherent mechanisms, and discovering drugs as well as having a therapeutic potential for the treatment of diseases. So, in this study, sensor systems based on Reflectometric Interference Spectroscopy (RIfS) have been prepared for selectively recognition and binding of TNF-alpha biomolecules. For this purpose, photosensitive nano structured TNF-alpha has been synthesized applying AmiNoAcid (monomer) Decorated and Light Underpining Conjugation Approach (ANADOLUCA) method using bis(2-2'-bipyridyl) MATyr-MATyr- ruthenium(II) (MATyr-Ru-MATyr) as a photosensitive monomer. Then, these photosensitive nano structured TNF-alpha have been used for TNF-alpha recognition as an alternative and unique sensor method. Also, the affinity constant of RIfS sensor has been calculated. The method has been showed high sensitivity, good precision and accuracy, and suited for the detection of TNF-alpha from aqueous solution. (C) 2013 Elsevier B.V. All rights reserved.