Surface-based biosensing devices benefit from a dedicated design of the probe layer present at the transducing interface. The layer architecture, its physicochemical properties, and the embedding of the receptor sites affect the probability of binding the analyte. Here, the enhancement of the probe density at the sensing interface by tuning the exponential growth regime of polyelectrolyte multilayers (PEMs) is presented. PEMs were made of poly-l-lysine (PLL), with appended clickable dibenzocyclooctyne (DBCO) groups and oligo(ethylene glycol) chains, and poly(styrene sulfonate) (PSS). The DNA probe loading and target hybridization efficiencies of the PEMs were evaluated as a function of the PLL layer number and the growth regime by a quartz crystal microbalance (QCM). An amplification factor of 25 in the target DNA detection was found for a 33-layer exponentially grown PEM compared to a monolayer. A Voigt-based model showed that DNA probe binding to the DBCO groups is more efficient in the open, exponentially grown films, while the hybridization efficiencies appeared to be high for all layer architectures. These results show the potential of such engineered gel-like structures to increase the detection of bio-relevant analytes in biosensing systems.