Efficient, sensitive, and convenient quantum dots (QDs) fluorescent immunosensors have a wide range of applications in the field of in vitro diagnostics (IVD). However, traditional single-signal readout methods are susceptible to interference, have limited sensitivity, and involve time-consuming detection processes. These have become key bottlenecks restricting their use in point-of-care (POC) detection. Herein, we developed a novel immunosensor based on QDs ratiometric fluorescence (QRF) and magnetic driven technology for sensitive, rapid, accurate detection of C-reactive protein (CRP). High-quality magnetic-fluorescent QDs nanobeads (QBs) were prepared by efficiently and orderly layered loading of magnetic nanoparticles (MNP) and red QDs using mesoporous silica nanoparticles (MSN) as a template. The magnetic driven-QRF-immunosensor was constructed based on green QDs probes-functionalized microplate. The results showed that the linear range of magnetic-fluorescence probes for the detection of CRP was 0.1-1,000 ng/mL, with a sensitivity of up to 0.05 ng/mL. Compared to conventional QRF-immunosensor, the detection time was halved (within 30 min), and the sensitivity improved by approximately 5-fold. Recovery experiments and clinical sample analyses also demonstrated the good accuracy of this immunoassay. Therefore, the constructed novel magnetic driven-QRF-immunosensor shows great potential for clinical diagnosis and disease monitoring, and is expected to be applied in POC detection as a new generation of detection technology.