The present work deals with the development of a Love-wave bacteria-based sensor platform for the detection of heavy metals in liquid medium. The acoustic delay-line is inserted in an oscillation loop in order to record the resonance frequency in real-time. A Polydimethylsiloxane (PDMS) chip with a liquid chamber is maintained by pressure above the acoustic wave propagation path. Bacteria (Escherichia coli) were fixed as bioreceptors onto the sensitive surface of the sensor coated with a polyelectrolyte (PE) multilayer using a simple and efficient layer-by-layer (LbL) electrostatic self-assembly procedure. Poly(allylamine hydrochloride) (PAH cation) and poly(styrene sulfonate) (PSS anion) were alternatively deposited so that the strong attraction between oppositely charged polyelectrolytes resulted in the formation of a (PAH-PSS)n-PAH molecular multilayer. The real-time characterization of PE multilayer and bacteria deposition is based on the measurement of the resonance frequency perturbation due to mass loading during material deposition. Real-time response to various concentrations of cadmium (Cd2+) and mercury (Hg2+) has been investigated. A detection limit as low as 10−12 mol/l has been achieved, above which the frequency increases gradually up to 10−3 mol/l, after a delay of 60 s subsequent to their introduction onto bacterial cell-based biosensors. Beyond a 10−3 mol/l a steep drop in frequency was observed. This response has been attributed to changes in viscoelastic properties, related to modifications in bacteria metabolism.