An ultrasensitive, fast and specific fluorescent platform for protein detection is developed. In this protocol, silver nanoparticles were conjugated with paramagnetic particles (MPs-Ag) for target capture, concentration and separation; fluorescent dyes functionalized silver nanoparticles (Tag) for generating signals. The presented method is highly sensitive and specific with a detection limit of 2.2 pM for thrombin, and no significant interference was observed for other proteins such as human serum albumin (HSA), lysozyme and IgG. This novel approach combining the magnetic separation and concentration of MPs-Ag, aptamer recognition and fluorescence enhancement of Tag, can be successfully used to enhance the sensitivity of detecting ultra-low levels of target proteins or biomolecules.