Tumor necrosis factor-alpha (TNF-α) is a signaling protein of inflammatory processes. TNF-α overexpression triggers inflammatory processes related to various diseases such as rheumatoid arthritis, Crohn’s disease, Chagas, and others. For this reason, the TNF-α has been used as an important biological biomarker for prognosis, understanding, and disease treatment. Surface plasmon resonance (SPR)-based biosensors are a good alternative for TNF-α detection because they are more sensitive than other techniques and have the ability to be developed miniaturized systems. Then we developed a nanohole array on gold nanofilm-based SPR biosensor for TNF-α detection. A biological biorecognition system (cysteamine/biotin/streptavidin/TNF-α antibody) was formed and TNF-α antigen was detected by transmitted light intensity monitoring. TNF-α antigen at 17  pg mL^−  1 was detected from fungus Paracoccidioides brasiliensis-infected rat blood serum. This concentration is far below those found in similar studies in the literature. This plasmonic device opens new opportunities for TNF-α detection.