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A rapid, label-free assay using photonic crystal biosensors for the direct detection of intact viable rotaviruses is demonstrated. This paper describes the design and operation of the biosensor, surface functionalization with anti-rotavirus antibody, characterization of the detection sensitivity, and demonstration of specificity with respect to non-target virus particles. A 30-min assay of a partially processed water sample yielded a detection sensitivity of 36 virus focus forming units without the use of any external reagents. This sensitivity is comparable to commercial enzyme-linked immunosorbent assays. These results suggest photonic crystal biosensors may be useful for real-time monitoring of virus contamination of environmental water resources.