A continuous-flow immunosensor for detection of trinitrotoluene (TNT) in aqueous solution is described. The sensor utilizes an immobilized monoclonal antibody specific for TNT and saturated with fluorescein-labeled antigen. When introduced into the flow stream through the column, samples containing unlabeled antigen displace the bound labeled antigen. The displaced labeled antigen is detected downstream using a fluorometer. The displacement of labeled antigen is directly proportional to the concentration of unlabeled antigen applied to the column over a range of 20 to 1200 ng/mL (ppb). The sensitivity is increased when solvents (ethanol or 2-propanol) are included in the flow buffer at concentrations up to 25% (v/v). By use of a cyclone air sampler and a TNT vapor generator, TNT could be detected in collected samples. The flow immunosensor provides the means to detect trace levels of TNT with high specificity in times of less than 1 min. Such qualities make the flow immunosensor a promising low-cost alternative to explosive detection devices currently available to transportation and security industries.