Owing to their unique optical properties, quantum dots (QDs) with different colors have been applied for simultaneous detection of multiple analytes. However, the use of single QD for multiplex detection of analytes with single-molecule detection has not been explored. Here we report a single QD-based nanosensor for multiplex detection of HIV-1 and HIV-2 at single-molecule level in a homogeneous format. In this single QD-based nanosensor, the QD functions not only as a fluorescence pair for coincidence detection and as a fluorescence-resonance-energy-transfer (FRET) donor for FRET detection but also as a local nanoconcentrator which significantly amplifies the coincidence-related fluorescence signals and the FRET signals. This single-QD-based nanosensor takes advantage of a simple 'mix and detection' assay with extremely low sample consumption, high sensitivity, and short analysis time and has the potential to be applied for rapid point-of-care testing, gene expression studies, high-throughput screening, and clinical diagnostics.