In this article, a novel dual-color quantum dot–encoded fluorescent nanoprobe was prepared by the reverse microemulsion method and layer-by-layer assembly method. First, red fluorescence–emitting CdTe quantum dots were encapsulated in silica nanoparticles by the reverse microemulsion method. Yellow fluorescence–emitting quantum dots were deposited on the surface of silica nanoparticles to form a dual-color quantum dot@silica beads/quantum dot nanoprobe. Then capture DNA was linked to a QSQ nanoprobe via covalent bonding. We utilized the quantum dot@silica beads/quantum dot nanoprobe to capture and detect the mutant BRAF DNA sequence through the competitive immunoassay method. The resulting quantum dot@silica beads/quantum dot nanoprobe-capture DNA conjugates showed sequence-specific hybridization with target DNA. Furthermore, a multispectral imaging system was utilized to distinguish the quantum dot optical code in the quantum dot@silica beads/quantum dot nanoprobe. The quantum dot@silica beads/quantum dot nanoprobe was used in human osteoblast-like HepG2 cell imaging. The proposed quantum dot@silica beads/quantum dot nanoprobe and decoding analysis method could be used for targeting imaging, biological assays, and early detection of cancer.