Application of polyelectrolyte multilayer (PEM) capsules as vehicles for the controlled delivery of substances, such as drugs, genes, pesticides, cosmetics, and foodstuffs, requires a sound understanding of the permeability of the capsules. We report the results of a detailed investigation into probing capsule permeability via a molecular beacon (MB) approach. This method involves preparing MB-functionalized bimodal mesoporous silica (BMSMB) particles, encapsulating the BMSMB particles within the PEM film to be probed, and then incubating the encapsulated BMSMB particles with DNA target sequences of different lengths. Permeation of the DNA targets through the capsule shell causes the immobilized MBs to open due to hybridization of the DNA targets with the complementary loop region of the MBs, resulting in an increase in the MB fluorescence. The assay conditions (BMSMB particle concentration, MB loading within the BMS particles, DNA target concentration, DNA target size, pH, sodium chloride concentration) where the MB-DNA sensing process is effective were first examined. The permeability of DNA through poly(sodium 4-styrenesulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) multilayer films, with and without a poly(ethyleneimine) (PEI) precursor layer, was then investigated. The permeation of the DNA targets decreases considerably as the thickness of the PEM film encapsulating the BMSMB particles increases. Furthermore, the presence of a PEI precursor layer gives rise to less permeable PSS/PAH multilayers. The diffusion coefficients calculated for the DNA targets through the PEM capsules range from 10-19 to 10-18 m 2 s-1. This investigation demonstrates that the MB approach to measuring permeability is an important new tool for the characterization of PEM capsules and is expected to be applicable for probing the permeability of other systems, such as membranes, liposomes, and emulsions.