A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ∼3×5 in2 surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ = 632.8 nm laser (cyanine-5) and a dye excited by a λ = 532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32x increase in fluorescent signal intensity for cyanine-5 conjugated strepavidin labeling, while a 25x increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm < λ < 633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays.