This work demonstrated a sensitive but direct method in detecting cancer biomarkers using dark field microscopy (DFM). Prostate-specific antigen (PSA), a well-known biomarker for prostate cancer, was chosen as the analyte to prove the concept. The immunoassay-based detection was performed in glass flow cell. Antibody-conjugated silver nanoparticles (AgNP-Ab) were served as the probe to capture PSA and then formed AgNP-Ab-PSA complexes on the surface of flow cell. The number of complexes is corresponding to the amount of PSA, which can be quantified by nanoparticle counting with DFM coupled with charge-coupled device (CCD) camera. The detection limit at 9 pM of this assay is well below the PSA threshold of prostate cancer patient, suggested the feasibility of our assay in diagnosis application. © 2013 Elsevier B.V.