In this study, a new detector for multiwavelength emission analytical ultracentrifugation (MWE-AUC) is presented, which allows measuring size- or composition-dependent fluorescence properties of nanoparticle ensembles. Validation of the new setup is carried out via comparison to a benchtop photoluminescence spectrometer and the established extinction-based multiwavelength analytical ultracentrifuge (MWL-AUC). The results on fluorescent proteins and silica particles demonstrate that the new device not only correctly reproduces sedimentation and diffusion coefficients of the particles but provides also meaningful fluorescence spectra. As an application example for a sample exhibiting a broad particle size distribution, spectra and size of graphene oxide nanoplatelets are extracted simultaneously. Narrowly distributed CdSe/ZnS quantum dots showing size- and structure-dependent shifts of their fluorescence spectra are analyzed as well. The combination of MWE- and MWL-AUC provides a comprehensive framework for the optical characterization for nanoparticles and macromolecules in terms of their extinction and emission properties.