Advances in nanoparticle technology have recently offered new tools to the bioanalytical field of research. In particular, new nanoparticle-based sensors have appeared able to give quantitative information about different species (ions, metabolites, biomolecules) in biosamples through ratiometric measurements. This article describes the methodologies developed so far in the design of such nanosensors. In particular, the different approaches to immobilize fluorescent chemosensor dyes to nanoparticles are presented. Concept designs of ratiometric nanosensors in terms of composition and architecture are also described and illustrated with examples taken from the literature.