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We have developed a multifocal diffractive optical fluorescence correlation spectroscopy system for parallel excitation and detection of single tetramethylrhodamine (TMR-4-dUTP) biomolecules in microarrays (Gene chips). Multifocal excitation was made possible through the use of a 2 × 2 fan-out diffractive optical element (DOE or kinoform) with uniform intensity in all four foci. Results of parallel excitation and detection in a high-density microarray of circular wells (o 70 μm), using the multifocal diffractive optical element designed 2 × 2 fan-out, show single biomolecule (TMR-4-dUTP) sensitivity in all foci, simultaneously. Future drug screening or functional genome analysis can hereby profit from this high sensitivity, parallel, fluorescence detection principle in biochip microarrays.