A novel and robust enzymatic biosensing platform with high sensitivity is developed based on facile one-step assembled bio-nanocomposites with enzymes-loaded polymeric nanoparticles decorating multi-walled carbon nanotubes (MWCNTs). An amphiphilic copolymer PAVE containing photo-cross-linkable coumarin segments and carboxylic groups was co-assembled with MWCNTs in aqueous solution while encapsulating the model enzyme namely glucose oxidase (GOx) simultaneously, generating necklace-like bio-nanocomposites (GOx@PAVE-CNTs) with GOx-loading polymeric nanoparticles as nanobeads and MWCNTs as conducting micron-string. Then the GOx@PAVE-CNTs bio-nanocomposites were electro-deposited onto electrode surface and a robust biosensing complex film with porous network structure was formed after following photo-cross-linking. Consequently, an enzymatic glucose biosensor was successfully constructed. The biosensor exhibited ultrafast response (<3 s) to glucose with a considerably wide linear range (1.0 μM ∼ 5 mM) and a low detection limit (0.36 μM) for glucose detection. High sensitivity and selectivity of the biosensor toward glucose were also well demonstrated. Furthermore, the biosensor showed exceptionally good stability and reproducibility. More importantly, the glucose biosensor was practically used for glucose detection from human urine and serum samples with satisfactory results. As a proof-of-concept strategy, this facile and effective strategy for biosensor fabrication is of considerable interest because of its versatility to be generalized to many other enzymatic biosensor systems, exhibiting promising and practical potential in bio-medical and life health applications.