We present a detailed description of a laboratory-based multispectral fluorescence imaging system (MFIS) for plant leaves. Fluorescence emissions with 360-nm excitation are captured at four spectral bands in the blue, green, red, and far-red regions of the spectrum centered at 450, 550, 680, and 740 nm, respectively. Preliminary experiments conducted with soybean leaves treated with a herbicide (DCMU) and short-term exposures to moderately elevated tropospheric ozone environment demonstrated the utilities of the newly developed MFIS. In addition, with the aid of fluorescence images of normal soybean leaves, several mechanisms governing the fluorescence emissions are discussed. Imaging results illustrate the versatility of fluorescence imaging, which provides information on the spatial variability of fluorescence patterns over leaf samples.