A fiber optic immunosensor has been developed for simultaneous detection of the most common explosives, 2,4,6, trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It employs a competitive immunoassay on the antibody-coated fiber optic probes, in which a fluorescent antigen competes with free antigen of unknown concentration for binding sites on the fiber surface. To achieve dual explosive detection, two alpha-TNT fiber probes and two alpha-RDX fiber probes are connected in series. The sample is mixed with fluorescent analogs, Cy5-ethylenediamine-trinitrobenzene (Cy5-EDA-TNB) and Cy5-ethylenediamine-RDX hapten (Cy5-EDA-RDH). Inhibition of the maximum signal in the presence of the sample is proportional to the concentration of the explosive. The detection limits for the multi-analyte assays are equivalent (6 ng/ml for both TNT and RDX) to those of the individual assays (5 ng/ml for both TNT and RDX). The standard curves for TNT and RDX represent a Linear relationship between percent signal inhibition and the natural logarithm of analyte concentration in the multi-analyte format, as well as in single analyte assays, thus allowing a simple and precise method of quantification. There is minimal cross-reactivity for the two antigens in the multi-analyte immunosensor, so it is also an effective means in analyzing samples containing mixtures of RDX and TM: (C) 1999 Elsevier Science B.V. All rights reserved.