A variety of particles have been adopted as solid support in bioaffinity assays, and the ability to modify the particle properties makes them a versatile tool in assay development. In separation-free fluorescence resonance energy transfer (FRET) assays, fluorescent particles have been used as donors and acceptors due to their high binding capacity and high specific activity. The availability of multiple fluorophores within the FRET range renders the orientation of individual donors and acceptors a non-factor enabling efficient sensitization. The high specific activity, being proportional to the particle size, may also inflict background fluorescence due to distance dependency of FRET, if the donor emission and detection wavelengths overlap. This is the first study to evaluate the impact of differently sized (47, 68, 92 and 202 nm in diameter) europium(III) chelate-doped polystyrene/acrylic acid donor particles on the core-related background fluorescence in a separation-free FRET-based assay utilizing competitive adsorption of acceptor-labelled protein. Within the particle size range studied, the particle core size-related background fluorescence showed no significant effect on the assay performance at the detection wavelength, which was selected on account of donor emission minimum. The assay sensitivity and dynamics showed compliancy with the rules of competitive assay. The results can be used in designing a competitive separation-free FRET-based assay utilizing particles as donors. In accordance, we applied the method for protein quantification. Sample protein prevented the adsorption of acceptor-labelled protein on the nanoparticle reducing energy transfer. Using this approach, a 100- to 1000-fold lower protein concentration was measured in comparison to traditional photometric protein assays suggesting that a high sensitivity assay can be constructed using FRET-based nanoparticle assay concepts.