We report a novel and very simple method for the detection of proteolytically-active prostate specific antigen (PSA) at a very low concentration using a self-assembled monolayer (SAM) of magnetic beads conjugated with peptide. The application of surface plasmon resonance (SPR) allows real-time optical characterization of the constructed recognition monolayer. This set-up is also capable of rapid monitoring of PSA samples through an instantaneous detection of medium refractive index shift induced by the enzymatic cleavage of the peptide and the dissociation of free magnetic beads. The results showed the ability of our SPR sensor to detect PSA within a short time (few minutes to 30 min) with high sensitivity (100 pg/ml) and specificity. Thanks to the elimination of washing and blocking steps, as well as the signal amplification and labeling procedures, this approach can be implemented in other miniaturized configurations such as screen printed electrode and biochip array amenable to low-cost point-of-care devices.