A magnetic bead based sensitive sandwich assay for C-reactive protein (CRP) has been developed. In this assay, magnetic microbeads conjugated with monoclonal anti-CRP are used to capture CRP in human serum, and then sequentially incubated with biotinylated monoclonal anti-CRP and streptavidin coated quantum dots (QDs) to form sandwich immunocomplexes. The immunocomplexes are further mixed with an immunoaffinity separation buffer to release QDs from the magnetic bead surface by dissociating antibody–antigen pairs. After magnetic bead separation, the fluorescence signal of QDs released in the immunoaffinity separation buffer is measured to quantify CRP. This proposed approach takes advantages of magnetic beads for fast magnetic separation, sandwich binding of two monoclonal antibodies for high specificity, and QD fluorescence labeling for high detection sensitivity. Moreover, it adopts immunoaffinity separation to avoid the interference of magnetic bead autofluorescence in signal transduction and thus enhances the detection resolution of the assay. The detection limit of CRP is 0.5 fM, corresponding to 10 pM CRP in 50 μL of sample volume, and the detection range is of around 3 orders of magnitude.