Honeybees are vital for biodiversity and agricultural productivity, yet their populations are declining globally, partly due to exposure to neonicotinoid pesticides. Odorant-binding protein 14 (OBP14) plays an important role in honeybee chemosensation, but its involvement in neonicotinoid toxicity remains underexplored due to limitations in traditional fluorescence spectroscopy techniques. This gap hampers our understanding of neonicotinoid risks to honeybee health. Here, we explored the molecular interactions between OBP14 from Apis mellifera and three widely used neonicotinoids (imidacloprid, thiamethoxam, and clothianidin) using molecular modeling, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and multispectroscopy. SPR and ITC characterized the binding affinity, specificity, and thermodynamic parameters of AmelOBP14 interacting with three neonicotinoid compounds, revealing that the binding process is spontaneous and primarily driven by hydrophobic and electrostatic interactions. Molecular modeling highlighted that phenylalanine residue Phe54, near the binding site, plays a critical role in these interactions. UV-vis absorption spectroscopy and synchronous fluorescence spectroscopy (SFS) support slight changes in the microenvironment around the aromatic amino acids of OBP14. Fourier Transform Infrared Spectroscopy (FTIR) and circular dichroism spectroscopy (CD) indicate a decrease in the α-helix content of OBP14, suggesting a change in its secondary structure, while three-dimensional (3D) fluorescence spectroscopy confirms the non-fluorescent nature of the OBP14 polypeptide backbone. The study results revealed its potential as a biomarker for pesticide risk assessment, providing important insights into the molecular mechanisms by which neonicotinoids may impair bee chemosensory function, and offering guidance for the design of safer pesticides to minimize harm to these important pollinators.