A growth-chamber experiment was conducted to evaluate whether ethylenenediurea (EDU), a chemical shown to be protective against ozone pollution, could ameliorate foliar damage induced by ultraviolet-B (UV-B) radiation exposure in ‘Roanoke’ soybean (Glycine max L.), a UV-B–sensitive cultivar, and whether these effects could be discriminated using fluorescence (F) observations. The experiment had four treatment groups: control; biologically effective UV-B (18 kjm−2 day−1); EDU (500 μmol mol−1); and both UV-B and EDU (UV/EDU). Measurements included photosynthetic pigments, F image system (FIS) images of adaxial surfaces in four spectral regions (blue, green, red and far-red) and F emission spectra of the pigment extracts produced at two excitation wavelengths, 280 nm (280EX) and 380 nm (380EX). Several F ratios from 280EX, 380EX and the FIS images successfully separated the low UV vs high EDU group responses based on means alone, with intermediate values for controls and the combined UV/EDU groups. A UV-B/blue emission ratio, F315/F420 (280EX), was correlated with chlorophyll content (μg cm−2)(R= 0.88, P < 0.001), as was a ratio of emissions at two UV-A wavelengths: F330/F385 (280EX) (R= 0.87). These two 280EX ratios were also linearly correlated with emission ratios produced by 380EX, such as the far-red/green ratio, F730/F525 (380EX) (R= 0.92, P < 0.001), and clearly distinguished the UV-B and EDU groups separately, and which bracketed the similar intermediate responses of the UV/EDU and control groups. The FIS images additionally captured the following anatomical spatial patterns across the leaf surfaces: (1) emissions of UV-B–irradiated leaves were more uniform but lower in intensity than those of other groups; and (2) emissions of EDU-treated leaves exhibited the greatest variation in spatial patterns because veins had elevated blue F and leaf edges had enhanced red and far-red F. This experiment supports the hypothesis that EDU substantially ameliorated UV-B damage to foliage, a result that relied on the combined use of FIS images and emission spectra.