In the present study, we report a novel separation-free method to detect and quantify avian influenza virus A (H5N1) nucleic acid without amplification, based on the alteration of photophysical parameters of quantum dot (QD) probes after hybridization with specific complementary target DNA. The target DNA was quantified in a custom-made portable device by simultaneously measuring lifetime and quenching of the QD probes. QD probes (25-mer) showed a 30% lifetime reduction and 40% fluorescence quenching when hybridized with complementary 25-mer target DNA. In comparison with a conventional QD-based assay, this assay provides a simple quantitation of nucleic acids with a single labeling step.