In this study, we investigated the detection limit of a competitive immunoassay using a surface plasmon resonance (SPR) sensor by both experimental and theoretical approaches. Highly sensitive explosive detection is required to prevent damage from hidden explosives such as improvised explosive devices (IED), and therefore in this study we focused on trinitrotoluene (TNT) detection using an anti-TNT antibody. As an experimental approach, competitive immunoassay experiments were conducted using a published technique for TNT detection, and the results were statistically analyzed to estimate the detection limit according to a reported method for an enzyme-linked immunosorbent assay (ELISA). In addition, a mathematical model describing measurement errors was developed and the detection limit was determined based on the correlated data between the SPR sensor response and antibody binding obtained from the experiments. Both approaches showed that the detection limit is about 10 ppb (ng/mL), and hence the theory is in good agreement with the experiment. Finally, using our mathematical model, we proposed a method for determining the detection limit in the presence of environmental influences.