Real-time, single particle fluorescence instruments used to detect atmospheric bioaerosol particles are increasingly common, yet no standard fluorescence calibration method exists for this technique. This limits the utility of these instruments as quantitative tools and complicates comparisons between different measurement campaigns. To address this need we have developed a method to produce size-selected particles with a known mass of fluorophore, which we use to calibrate the fluorescence detection of a Wide-band Integrated Bioaerosol Sensor (WIBS-4A). We use mixed tryptophan-ammonium sulfate particles to calibrate one detector (FL1; excitation = 280 nm; emission = 310–400 nm), and pure quinine particles to calibrate the other (FL2; excitation = 280 nm; emission = 420–650 nm). This procedure allows users to set the detector gains to achieve a known absolute response, calculate the limits of detection for a given instrument, improve repeatability of instrumental set- up, and facilitate intercomparisons between different instruments. We recommend calibration of single-particle fluorescence instruments using these methods.