Fluorescence polarization (FP) is attractive for measuring binding interactions and has been recently used to study DNA adsorption on nanomaterials. Since most nanomaterials are strong fluorescence quenchers, correlations among adsorption efficiency, quenching efficiency, and FP need to be interpreted carefully. In this work, carboxyfluorescein (FAM)-labeled DNA oligonucleotides were studied under various quenching conditions. First, quenching was induced by lowering the pH, taking advantage of the fact that FAM is almost nonfluorescent at a pH below 4. Strong interactions were observed between the FAM label and polyadenine DNA, as judged by the increased FP at low pH, while FAM-labeled polythymine DNA was less affected by the pH. Comparisons were also performed with FAM-labeled poly(ethylene glycol) and bovine serum albumin. An equation was derived to calculate the effect of fluorescence quenching and DNA adsorption by nanomaterials. For strongly quenching nanomaterials, such as graphene oxide, DNA adsorption alone does not change the measured FP. Light scattering and weak fluorescence from graphene oxide increase FP in these cases. For comparison, a strongly adsorbing but weak quenching material, Y2O3, was also studied and the result was consistent with a normal binding reaction. Overall, FP is a powerful technique for binding and adsorption assays, but quenched samples need to be interpreted with care.