Au and Ag nanoparticles (NPs) have been known to display significantly enhanced two-photon photoluminescence (2PPL) upon the formation of nanoparticle aggregates. The enhancement effect of the core-shell nanoparticles has not been explored so far. Here we have prepared Au@Ag bimetallic core-shell nanoparticles with different thicknesses (1.1, 2.1, 3.5, 4.5, and 5.5 nm) of silver coating on 19 nm Au NPs to investigate the composition effects on plasmon coupling-enhanced 2PPL. A maximum 2PPL enhancement factor (IcoupledNPs/IisolatedNPs) of up to 840-fold was obtained for Au@Ag NPs with ∼3.5 nm Ag nanoshells. These Au@Ag NPs were subsequently utilized in two-photon detection of S1 nuclease as a photoluminescence turn on probe. This method displayed high sensitivity with the limit of detection of 1.4 × 10(-6) U μL(-1) and an excellent selectivity.