A series of room-temperature ionic liquids (RTILs) containing different functional groups such as hydroxyl, nitrile, carboxyl, and thiol attached to imidazolium cations, combined with various anions such as chloride [Cl], tetrafluoroborate [BF(4)], hexafluorophosphate [PF(6)], and bis[(trifluoromethyl)sulfonyl]imide [Tf(2)N], have been successfully synthesized. Dissolved in chitosan (Chi), the Chi/RTIL composites can be employed as flexible templates for the preparation of Au/Pt nanostructures. These Au/Pt nanostructures can be facilely deposited in situ on the surface of Chi/RTILs through electrodeposition. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) results demonstrate that the alloy size is significantly dependent on the structure of the Chi/RTILs, with sizes ranging from 2.8 to 84.7 nm. Based upon the functionalized RTILs, nine Chi/RTIL-Au/Pt biosensors have been fabricated. First, the size-dependent electrochemistry of Chi/RTIL-Au/Pt was investigated using potassium ferricyanide as the probe. The reversible electron transfer of the Fe(CN)(6)(3-/4-) redox couple was realized for the nine biosensors, and the peak currents, as well as the peak-to-peak separations (ΔE(p)) and electron-transfer rates, differ greatly from each other because of the diversity of the RTILs. Further electrochemical research reveals that the functional groups of these RTILs exert an evident influence on the reduction behavior of H(2)O(2), which in turn illustrates that the electrocatalytic activity of Chi/RTIL-Au/Pt nanocomposites can be tuned by means of employing RTILs with different functional groups, and an appropriate combination of cations and anions may produce a higher activity. The facilitated electron transfer and the intrinsic catalytic activity of Au/Pt NPs provide a facile way to construct a third-generation H(2)O(2) biosensor with a high sensitivity, low detection limit, quick response time, and excellent selectivity.