A real-time light-emitting-diode-induced fluorescence biological aerosol detection method is developed that enables real-time monitoring of airborne particles derived from organisms so the presence of these contaminants can be identified. To characterize the performance, a variety of background measurements describing different contamination levels and particulate concentrations controlled to closed environments were obtained and used as the input to a detection algorithm. Optimization was performed for various backgrounds. Additionally, detection performance per sensitivity level was determined using a confusion matrix so that the user can select sensitivities depending on the background. For 50 to 100 agent-containing-particles-per-liter-of-air target detection performance, an alarm rate of 100% was achieved, and the false-alarm rate was optimized to 0%. Applying these settings, the final sensitivities of the “high” and “low” modes were roughly twice and half of those of the “normal” mode, respectively. The final alarm thresholds of high and low sensitivities were set to 0.1 times and 10 times the reference values, respectively, enabling reliable operation in a variety of situations.