Tumor necrosis factor α (TNFα) is an inflammatory cytokine that is involved in immune function and is proposed to play a role in metabolic disorders. Although some bovine-specific methods have been published recently, assays used for determining plasma TNFα concentration in bovine disease models often do not offer acceptable precision for measurement of basal concentrations in healthy animals. The objective of this work was to develop an effective, low-cost sandwich ELISA procedure with improved sensitivity. A protocol developed for use with cell culture supernatant was modified for use with bovine plasma and serum by optimizing antibody concentrations, incubation times and temperatures, and standard diluents. The coating antibody concentration was decreased from 10 to 6.8 μg/mL, whereas the detection antibody concentration remained 2.5 μg/mL. Sample incubation was increased from 1h at room temperature to an overnight incubation at 4°C, which increased the sensitivity of the assay. Multiple matrices were tested for dilution of standards and were assessed by determining recovery of bovine TNFα spiked into bovine serum and plasma. Recoveries were acceptable in both bovine serum and plasma (71-103%) when quantified with standards diluted in human serum or phosphate-buffered saline. The modified bovine TNFα ELISA offers a detection range of 2 to 250 pg/mL. This detection limit is at least an order of magnitude lower than previously reported, and will allow for greater precision in determining basal TNFα concentrations in bovine plasma. The improved sensitivity of this ELISA will be critical to assessing current hypotheses concerning the metabolic effects of moderately elevated TNFα concentrations.