A surface plasmon resonance (SPR) biosensor, which contains an overlayer of titanium dioxide nanoparticles (TDNPs) to modify the plasmonic interface, has been developed and investigated. Owing to its large surface area and high refractive index, the TDNP overlayer significantly enhances the probing electric field intensity and detection sensitivity. This sensitivity is related to the TDNP overlayer thickness, which can be engineered by changing the TiO2-ethanol dispersion's spin-coating concentration. The highest refractive index sensitivity for ethylene glycol measurement is 2567.3 nm/RIU, which is 38% higher than that of a conventional SPR sensor with an uncoated gold film. The proposed TDNP-SPR sensor also exhibits a 1.59-fold sensitivity enhancement in fetal bovine serum detection. Moreover, the proposed interface modification approach that is applied without additional biochemical amplification steps is chemical-free and contamination-free; therefore this TDNP-SPR sensor could be integrated into a sensitive, cost-effective, and biocompatible platform for rapid and label-free biochemical detection.