A rapid nanochannel-based immunoassay capable of the filtering and subsequent detection of proteins in whole blood without any sample preparation is described. This is accomplished by using a nanoporous/nanochannel membrane modified with antibodies, the conductivity of which toward a redox indicator is tuned by primary and secondary immunoreactions with proteins and gold nanoparticles. This interesting nanopore blockage by gold nanoparticles is enhanced by silver deposition that further decreases the diffusion of the signaling indicator through the nanochannel. The efficiency of the nanochannels to act as immunoreaction platforms including the use of nanoparticles is also monitored by microscopic techniques. Successful detection of immunoglobulins including a cancer biomarker is achieved in buffer as well as in whole blood. This system constitutes an efficient immunoassay capable of detecting up to 52 U mL−1 of CA15-3. The developed nanochannel/nanoparticle-based device can be used for several other proteins and extended also to DNA detection with interest not only for diagnostics but also environmental monitoring, food analysis, safety, and security applications.