Objectives Alzheimer's disease (AD) is the most common cause of dementia in the world. As many AD biomarkers occur at rather low abundances in CSF or blood, techniques of very high sensitivity and accuracy are important as diagnostic tools in the clinic. Here, we aimed to provide proof of concept of the use of a single molecule detection technique, Fluorescence Correlation Spectroscopy (FCS) for detection of novel candidate biomarkers for AD. Design and methods FCS detects the diffusion times of the antigen-antibody complexes in highly diluted sample solutions, thus eliminating the need of large sample volumes and allows estimating the concentration of the target antigen. We developed a FCS set-up for contactin-2, a neuronal cell adhesion molecule and a ligand of beta-secretase 1 (BACE1) and amyloid precursor protein (APP), the latter proteins being important players in AD. With this method, we investigated whether contactin-2 concentrations are changed after delayed storage and in patients with Alzheimer's disease. Results The FCS set-up for measuring contactin-2 in CSF had a lower limit of quantification (LLOQ) of 0.2 ng/ml and intra- and inter-assay coefficients of variation (CVs) of 12.2% and 14.6% respectively. Contactin-2 levels were stable up to one week storage of CSF (n = 3) at RT and 4 °C. Further, contactin-2 levels were similar in probable AD patients (n = 34, p = 0.27) compared to patients with subjective cognitive decline (SCD) (n = 11). Conclusions FCS is a sensitive tool, which can be used for detecting biomarkers in the clinical setting using very low sample volumes (10 μl) and can measure proteins in their native conformations in the body fluid.