The increasing accessibility of 3D printers makes their use for criminal activity more likely. Current forensic analysis of trace evidence left by 3D-printed materials focuses on identifying the general type of plastic, which includes acrylonitrile butadiene styrene, polylactic acid, nylon, polycarbonate, polyethylene terephthalate, and chlorinated polyethylene. Herein, we present a nondestructive approach capable of differentiating among different types of nylons. The new approach is based on roomtemperature fluorescence spectroscopy. Excitation−emission matrices, excitation and emission spectra, and synchronous fluorescence spectra are directly recorded from single microplastics with the aid of a fiber-optic probe coupled to a commercial spectrofluorometer. The comparison of spectral features demonstrates the capability to differentiate microparticles originating from Nylon 11, Nylon 12, Nylon 6/6, and Nylon 6/12. The observed differences are attributed to the presence of fluorescent impurities embedded in the polymer during its fabrication. The outstanding matching of excitation−emission matrices, excitation and emission spectra, and synchronous fluorescence spectra demonstrates the potential of this approach to link trace evidence to a specific source beyond its general plastic type.