Quantum dot composite colloids (QDCCs), submicron-sized colloidal particles incorporating multiple QDs, have been employed as signal reporters. Multiple QDs were encapsulated within the hydrophobic pockets of amphiphilic polyethylenimine derivative (amPEI), forming amPEI-QDCCs with a hydrodynamic size of approximately 100 nm. Fluorescence (FL) correlation spectroscopy revealed that each QDCC encapsulates an average of 20 QDs. A layer-by-layer (LbL) multilayer shell composed of poly(sodium 4-styrenesulfonate) and polyethylenimine polyelectrolytes was added to amPEI QDCCs to improve surface functionalization and structural robustness. Increasing the number of LbL layers reduced copper ion (Cu²⁺) intrusion and prevented payload exchange of hydrophobic dark quencher in polymersomes, owing to strong hydration and improved shell integrity. The enhanced robustness allowed functionalization of the outer QDCC shell with amines, carboxylates, zwitterionic (ZW) moieties, or their combinations. ZW-functionalized QDCCs (ZW-QDCCs) exhibited colloidal stability across a wide pH range (pH 5-10) and in saturated NaCl solutions. Streptavidin (SA)/biotin-conjugated ZW-QDCCs exhibited specific labeling capabilities in bead assays. A sensitive and rapid enzyme-free FL immunoassay for C-reactive protein (CRP) was developed using sandwich-type immunoassay procedures and the biological self-assembly of the SA/biotin-ZW-QDCC pair. The high multivalency of SA or biotin, combined with the FL intensity of QDCCs, enabled a detection limit of 15.9 fM (1.91 ng/L) within 6 min for 6 amplification rounds─nearly 5 orders of magnitude lower than conventional CRP immunoassays. Our QDCCs demonstrate significant potential for use in ultrasensitive quantification platforms.