The efficient detection of tumor markers is crucial to early cancer diagnosis and monitoring. Here, we propose a unique “two birds with one stone” dissolution-enhanced afterglow bioassay based on amino-functionalized mesoporous SiO₂ nanoparticles (MSNs-NH₂) encapsulated Eu(OH)CO₃ nanoprobes. These nanoprobes (i) stably conjugate to the antibody and (ii) are highly soluble in the acidic enhancer solution, and provide a high Eu³⁺ concentration. The released Eu³⁺ ions further activate the photochemical afterglow system to determine the alpha-fetoprotein (AFP) levels in clinical serum samples. Without the use of a real-time excitation source, the interference of autofluorescence and scattering from blood can be avoided. The demonstrated limit of detection (LOD) for AFP was as low as 0.12 ng·mL⁻¹. This covalently connected dissolution-enhanced luminescent bioassay (DELBA) and novel afterglow system shows tremendous potential for ultrasensitive and rapid clinical diagnosis.