Honeybees, essential pollinators for maintaining biodiversity, are experiencing a sharp population decline, which has become a pressing environmental concern. Among the factors implicated in this decline, neonicotinoid pesticides, particularly those belonging to the fourth generation, have been the focus of extensive scrutiny due to their potential risks to honeybees. This study investigates the molecular basis of these risks by examining the binding interactions between Apis mellifera L. chemosensory protein 3 (AmelCSP3) and neonicotinoids with a cis-oxygen bridge heterocyclic structure. Employing surface plasmon resonance (SPR) in conjunction with multispectral techniques and molecular modeling, this study meticulously analyzed the binding affinity, specificity, and kinetics under conditions that simulate real-world exposure scenarios. Key parameters such as the number of binding sites (n), binding constants (K