Development of sensitive homogeneous assays is a high-priority research target for clinical diagnostics. Quantum dots (QDs) present favorable photophysical properties, which implies their potential as an exceptional dye in fluorescence detection. QDs-based biosensors have been described in the literature; however, few of them have truly progressed to widespread clinical usage. In this work, a chemiluminescent homogeneous detecting biosensor is fabricated using QDs-doped polystyrene nanospheres to sensitively detect biomarkers in low-volume serum samples. Phthalocyanine-dyed and QDs-encapsulated carboxylate-functionalized polystyrene nanospheres with surface carboxyl groups (PPs and QPs, respectively) were fabricated and served as triggers and fluorescent probes, respectively, in this biosensing system. In this sandwich-format immunoassay, the PPs produced singlet oxygen once sensitized by 680 nm diode lasers, and the QPs, conjugated with antibodies, and then reacted with the singlet oxygen in the presence of specific antigens and emitted anti-Stokes fluorescence with wavelengths around 605 nm, as a result of fluorescence resonance energy transfer (FRET) within the QPs. We demonstrated the determination of carcinoembryonic antigen as a model protein target in 25 μL of serum samples with an unprecedented detection limit of 2.56 × 10-13 M (46 pg/mL) using this biosensor. Furthermore, excellent correlations ( R2 = 0.99718, n = 107) were obtained between utilizing this biosensor and commercialized chemiluminescence immunoassay kits in clinical serum detection. These results demonstrate that our flexible and reliable biosensor is suitable for direct integration into clinical diagnostics, and it is expected to be a promising diagnostic tool for early detection and screening tests as well as prognosis evaluation for patients.