The detection of disease-related proteins in complex biological media demands sensitive, reliable, and low-cost sensing methods. We present here an assay with aptamer-functionalized silica nanoparticles (NPs) as the sensory platform for thrombin detection in blood serum. As a result of specific protein/aptamer interaction, the presence of thrombin induces the primary aptamer-functionalized NPs to form a sandwich structure with fluorescein-labeled secondary aptamer and results in fluorescent NPs. A conjugated polyelectrolyte is further added to the NP solution to amplify the fluorescent signal. The assay shows high specificity for thrombin detection in serum. In addition, a detection limit of 1.06 nM is obtained, which is significantly improved as compared to that for many fluorescence-based thrombin detection assays. Using lysozyme as a second example, the NP-based detection strategy is shown to be generic and could be readily applicable to the detection of other proteins in practical samples.