New polymer-enzyme-metallic nanoparticle composite films with a high-load and a high-activity of immobilized enzymes and obvious electrocatalysis/ nano-enhancement effects for biosensing of glucose and galactose are designed and prepared by a one-pot chemical pre-synthesis/ electropolymerization (CPSE) protocol. Dopamine (DA) as a reductant and a monomer, glucose oxidase (GOx) or galactose oxidase (GaOX) as the enzyme, and HA(u)Cl(4) or H2PtCl6 as an oxidant to trigger DA polymerization and the source of metallic nanoparticles, are mixed to yield polymeric.. bionanocomposites (PBNCs), which are then anchored on the electrode by electropolymerization of the remaining DA monomer. The prepared PBNC. material has good biocompatibility; a highly uniform dispersion of the nanoparticles with a narrow size distribution, and high load/activity of the immobilized enzymes, as verified by transmission/scanning electron microscopy and electrochemical quartz crystal microbalance. The thus-prepared enzyme electrodes show a largely improved amperometric biosensing performance, e.-g., a very high detection sensitivity (99 or 129 mu A cm(-2) mm(-1) for glucose for Pt PBNCs on bare or platinized Au), a sub-micromolar limit of detection for glucose, and an excellent durability, in comparison with those based on conventional procedures. Also, the PBNC-based enzyme electrodes work well in the second-generation biosensing mode. The proposed one-pot CPSE protocol may be extended to the preparation of many other functionalized PBNCs for wide applications.