A prototype bioluminescence-based biosensor was designed and constructed to evaluate the antimicrobial efficacy of chlorine dioxide (ClO2) gas under various treatment conditions. The biosensor consisted of a bioluminescent bioreporter (Pseudomonas fluorescens 5RL), an optical transducer (photomultiplier tube), and a light-tight chamber housing, the bioreporter and the transducer. The bioluminescent recombinant P. fluorescens 5RL in the biosensor allowed for online monitoring of bioluminescence during ClO2 gas disinfection. Experiments were performed to evaluate the effects of the two key physical parameters associated with ClO2 disinfection: relative humidity (40, 60, 80%) and ClO2 gas concentration (0.5, 1.0, 1.6, 2.1 mg/l) on the bioreporter. Results showed that increasing concentrations of ClO2 gas corresponded to a faster decrease in luminescence. The rates of luminescence decrease from P. fluorescens 5RL, and the log reduction time (LRT, time required to obtain 1-log reduction in luminescence) were calculated for each treatment tested. The LRT values of luminescence were 103, 78, 53, and 35 s for 0.5, 1.0, 1.6, and 2.1 mg/l of ClO2 gas treatment, respectively, at 78% relative humidity. The gas concentration which caused a tenfold change in LRT (z value) for luminescence of P. fluorescens 5RL was 3.4 mg/l of ClO2. The prototype biosensor showed potential for many applications, such as monitoring real-time microbial inactivation and understanding parameters that influence the efficacy of gaseous decontamination procedures.