Upconversion nanoparticles (UCNPs) are of high interest for biosensing because of their unique near‐infrared‐excitation and visible‐emission features. An emerging field within UCNP biosensing is the detection of biological interactions through Förster resonance energy transfer (FRET). However, the relatively large size, the distribution of emitting lanthanide ions within the nanoparticle, the unknown photoluminescence (PL) quantum yields (QY) of these emitting ions, and the many available core–shell architectures make the interpretation of UCNP‐based FRET data extremely difficult. Here, we present a detailed spectroscopic study of three types of NaGdF 4 :Er 3+ ,Yb 3+ UCNPs with and without shells and lanthanide‐ion doping in the cores or the shells. The different architectures strongly influence the brightness and PL lifetimes of the UCNPs, which are important properties for FRET to Cy3.5 dyes attached to the UCNP surfaces through DNA. Analysis of the FRET‐sensitized dye PL decays allows the determination of the FRET efficiencies, which, in turn, can be used to estimate donor–acceptor distances, Förster distances, and Er 3+ donor QYs, all of which are difficult to assess by other methods.
