The electrochemical glycerol oxidation reaction (GOR) offers a promising alternative to the anodic oxygen evolution reaction in water electrolysis, enabling simultaneous energy-saving hydrogen production and sustainable synthesis of value-added formate. Current advances reveal that high-valence transition metal species critically enhance C-C cleavage during glycerol electro-oxidation, with targeted enrichment of these active sites-via local structure design or in-situ reconstructionproving highly effective. Herein, we engineer a high-entropy FeCrCoNiCu layered hydroxide (HE-LH) catalyst featuring a hybrid quasi-single-crystalline (QSC)/amorphous nanostructure. This design synergistically integrates locally engineered and in-situ enriched high-valence active sites for efficient glycerol-to-formate conversion. The high-entropy composition induces a distinctive catalytic ensemble effect, elevating intrinsic GOR activity, while the QSC/amorphous heterostructure maximizes the density of electrochemically (re)active sites. Leveraging this dual optimization, HE-LH achieves an exceptional formate Faradaic efficiency of 92.9% and maintains >83% efficiency over five consecutive cycles. This work pioneers a co-design strategy for electrocatalysts by concurrently optimizing active site density and intrinsic activity, establishing high-entropy layered hydroxides as durable platforms for electrochemical biomass upgrading.