Guided-mode resonances enhanced excitation and extraction of two-photon photoluminescence (TPP) is demonstrated with a one-dimensional resonant waveguide grating (RWG) with a layer of fluorescent polymer (polyfluorene, PFO) on top. In this work, we design and fabricate a PFO RWG, in which two dispersive resonant modes in TE-polarization were measured. By aligning the red-shifting resonant mode with excitation wavelength in the infrared range, and the blue-shifting resonant mode with TPP spectrum in the visible range, the intensity of TPP can be enhanced up to 300-fold compared with that from a flat film with the same thickness coated on a glass slide. Such high enhancement results from firstly the strong evanescent local field in the waveguide layer due to the resonance between the incident light and the waveguide structure according to the results of rigorous coupled-wave analysis calculation, and secondly the enhanced extraction of the emission light which also resonates with the waveguide structure.