The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP−NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4‘-bipyridine (PPB) are described. The MBP domain of MBP−NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 × 104 and 7.1 × 104 M-1 s-1 were obtained for TNT and DNT, respectively. The apparent Michaelis−Menten constants (K M) for MBP−NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95 μM, respectively. The corresponding value for “wild-type” NR in solution containing TNT was 78 μM, which is very close to the value obtained for MBP−NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2 μM, and the sensitivities were determined to be 205 and 222 nA/μM, respectively.