The level of cancer biomarkers in cells, tissues or body fluids can be used for the prediction of the presence of cancer or can even indicate the stage of the disease. Alpha-fetoprotein (AFP) is the most commonly used biomarker for early screening and diagnosis of hepatocellular carcinoma (HCC). Here, a combination of three techniques (click chemistry, the biotinâstreptavidinâbiotin sandwich strategy and the use of antigenâantibody interactions) were combined to implement a sensitive fluorescent immunosensor for AFP detection. Three types of functionalized glasses (dibenzocyclooctyne- (DBCO-), thiol- and epoxy-terminated surfaces) were biotinylated by employing the respective adequate click chemistry counterparts (biotinâthiol or biotinâazide for the first class, biotinâmaleimide or biotinâDBCO for the second class and biotinâamine or biotinâthiol for the third class). The anti-AFP antibody was immobilized on the surfaces via a biotinâstreptavidinâbiotin sandwich technique. To evaluate the sensing performance of the differently prepared surfaces, fluorescently labeled AFP was spotted onto them via microchannel cantilever spotting (”CS). Based on the fluorescence measurements, the optimal microarray design was found and its sensitivity was determined.