Surface plasmon resonance (SPR) sensors are known to be able to detect very low surface concentrations of (bio)molecules on macroscopic areas. To explore the potential of SPR biosensors to achieve single-molecule detection, we have minimized the read-out area (to ~64 μm2) by employing a sensor system based on spectroscopy of surface plasmons generated on a diffractive structure via a microscope objective and light collection through a small aperture. This approach allows for decreasing the number of detected molecules by 3 orders of magnitude compared to state-of-the-art SPR sensors. A protein monolayer has been shown to produce a response of 5000 times the baseline noise, suggesting that as few as ~500 proteins could be detected by the sensor.