The molecular orientation of Cy5-labeled target DNA hybridized on a probe DNA layer was studied on a planar waveguide in the Kretchmarm configuration at various target DNA bulk concentration. Optical waveguide fluorescence spectroscopy (OWFS) was employed to evaluate the fluorescence emission anisotropy by analyzing two polarized modes, i.e., TM and TE modes propagating in a waveguide. In this way, the orientational order parameter S could be determined. It was found that the orientation of DNA duplex molecules is disordered at lower concentrations, while relatively ordered at higher concentrations.