Cyanine dyes have become widely used fluorescence labels in clinical and biological chemistry. In particular, cyanine dyes with excitation wavelengths lambda(ex) > 600 nm are often used in biological applications. However, aggregation behavior and matrix effects on cyanine fluorescence are not fully understood yet and interfere with the data interpretation. In this study, we analyzed the spectroscopic characteristics of a model system consisting of the biotinylated cyanine dyes DY-635 and DY-647 and their streptavidin conjugates. On the basis of the spectroscopic data, the interaction processes between cyanine dye molecules and proteins are discussed. Binding to streptavidin had a significant influence on both fluorescence and anisotropy decays of the cyanine dyes investigated. In particular, the fluorescence anisotropy was significantly altered, making it a promising detection parameter for bioanalytical applications in connection with the cyanine dyes used in the present study. In order to evaluate the time-resolved anisotropy, the introduction of a sophisticated kinetic model was required to describe the contributions from different fluorescing species properly. The rotational motion of streptavidin-bound dyes was analyzed using the associated anisotropy model, which allowed discrimination between contributions from different microenvironments. The anisotropy decay times increased by a factor of up to 20 due to protein binding.