A label-free localized surface plasmon resonance (LSPR)-based biosensor exploiting gold nanorods (GNRs) is proposed and demonstrated. For this aim, 35 ± 5 nm long and 20 ± 4 thick GNRs spaced by a few nanometers thick polyelectrolytes (PE) from a gold thin film was analyzed and synthesized. The morphology of the GNRs, the plasmon properties of GNRs, swelling of PE layers and the wettability of the surfaces were characterized by transmission and scanning electron microscopy, spectroscopic reflectivity and contact angle measurements, respectively. Indeed, when immersed in a phosphate buffer saline solution, the GNRs-PE-gold system shows an optical shift of the LSPR wavelength. This shift was found to correspond to a vertical swelling of about 2 nm, demonstrating the extreme sensitivity of the biosensor. Finally, we show that LSPR measurements can be used to detect dynamic resonance changes in response to both thickness and buffer solution, while the hydrophobic behavior of the surface can be exploited for reducing the number of liquid analytes in clinical biosensing application.