Aptamers are short, single-stranded RNA or DNA sequences, which can bind to protein ligands with high affinity and specificity. Applications of aptamers are broad, ranging from drugs and drug delivery vehicles to biosensors. Tumor necrosis factor (TNF) is an inflammatory cytokine that plays a critical role in the pathogenesis of several autoimmune inflammatory diseases. Blocking TNF activity by monoclonal antibodies or TNF receptor fusion protein has been tremendously successful in treating these diseases. However, manufacturing these biological TNF inhibitors is expensive and a significant proportion of patients do not respond to TNF blockade. Here we describe selection of single-stranded DNA aptamers for TNF blockage, and their bioactivity in blocking TNF-mediated cytotoxicity in vitro. These TNF-binding aptamers have the potential to serve as alternatives to biological TNF inhibitors and to be used as in vivo probes for TNF detection.