For the development of a quick and effective on-site sensor, a multifunctional composite with concentrating and sensing functions is greatly advantageous. The multifunctional composite with high magnetic susceptibility for concentration and enhanced fluorescence intensity for sensing is synthesized and applied for an on-site sensor. The multifunctional composite is prepared by incorporating a cluster of Fe3O4 nanocrystals at the centre and a QD-layer at radial equidistance near the surface of a silica microsphere (diameter ∼0.6 μm). The extinguished fluorescence of the composite, through hybridization with quenching DNAs via aptamers anchored on the composite, resumes instantaneously and quantitatively upon addition of target proteins by displacing the quenching DNAs. The configuration of thrombin binding aptamers and quenching DNAs on the composite is optimized to achieve a quenching efficiency of 94%. Upon simple injection of thrombin solution, binding of thrombin to its specific aptamer on the composite is detected at concentrations as low as 0.35 nM (10 ng mL-1) within 5 min. The current multifunctional composite system is expected to be a promising platform for on-site biosensors.