In this paper are the experimental results used to characterize four distinct monoclonal anti-TNT antibodies (in vivo and in vitro cloned) for potential use in a field-portable immunosensor. Direct and competitive enzyme-linked immunosorbent assays (ELISA) were performed to determine their affinity for TNT and a fluorescently labeled analog of TNT for use in an immunosensor. Effective concentrations (EC(50)), inhibition concentration (IC(50)) and cross-reactivity measurements to related nitroaromatics (e.g., 2,4,6-trinitrobenzene [TNB], methyl-2,4,6-trinitrophenyl nitramine [tetryl], 2-amino-4,6-dinitrotoluene [2A-4,6-DNT], 2,4-dinitrotoluene [2,4-DNT] and 1,3-dinitrotoluene [1,3-DNT]) were measured. Final characterization of the monoclonal antibodies was based on performance (measured by fluorescence dose response) using a fluorescence-based microcapillary displacement assay. Analytical techniques showed a high degree of affinity for TNT and varying degrees of cross-reactivity with each respective monoclonal antibody. Microcapillary displacement immunoassays with each of the antibodies resulted in detection capabilities at the lowest applied TNT concentration (10 ng/ml).