The surface plasmon-enhanced fluorescence measurement device has: a light source that irradiates the diffraction grating with a linearly polarized excitation light; a rotating part that changes the direction of the optical axis of the excitation light with respect to the diffraction grating when seen in plan view, or changes the polarization direction of the excitation light with respect to the diffraction grating; a polarizer that extracts linearly polarized light from the fluorescence emitted from the fluorescent substance; and a light detection unit that detects the linearly polarized light extracted by the polarizer.