We electrokinetically characterize properties of single 42-nm polystyrene nanoparticles (NP) in nanofluidic channels imaged with frustrated total internal reflection fluorescence microscopy (fTIRFM). Specifically, we demonstrate fTIRFM of individual NPs in nanofluidic channels shallower than the evanescent field and use the resultant illumination field to gain insight into the behavior and electrokinetic properties of individual NP transport in channels. We find that the electrophoretic mobility of nanoparticles in 100-nm channels is lower than in larger channels or in bulk, presumably due to hindrance effects. Furthermore, we notice a non-intuitive increase in mobility with buffer concentration, which we attribute to electric double layer interactions. Finally, since the evanescent field intensity decreases with distance from the channel wall, we use the measured fluorescence intensity to report probable transverse distributions of free-solution 42-nm polystyrene fluorescent particles. Our method promises to be useful for characterizing nanoscale molecules for many applications in drug discovery, bioanalytics, nanoparticle synthesis, viral targeting, and the basic science of understanding nanoparticle behavior.