In this work, we fabricated platinum nanoparticles/graphene nanohybrids (Pt/G) via in situ reduction of PtCl₆²⁻ in the presence of poly(diallyldimethylammonium chloride)-modified G, and used them as a support for the immobilization of horseradish peroxidase to construct a biosensor (HRP/Pt/G/glassy carbon (GC)) for H₂O₂. Poly(diallyldimethylammonium chloride) (PDDA), a positively charged polyelectrolyte, created a net positive charge on the carbon atoms in the all-carbon graphene plane via intermolecular charge transfer. Then the resultant positively charged PDDA-modified G was used for sequential self-assembly with negatively charged PtCl₆²⁻ which could be reduced in situ. The Pt/G hybrid provides an effective atmosphere for maintaining the bioactivity of HRP, and also acts as a bridge for the transfer of electrons between the active center of HRP and the electrode surface, thereby resulting in a more rapid transmission of electrons. The HRP/Pt/G/GC showed high sensitivity and fast response for electrocatalytic reduction towards H₂O₂, revealing that Pt/G effectively reinforced the immobilization of HRP and enhanced the utilization of HRP. The linear range for H₂O₂ was estimated to be from 3.0 × 10⁻⁶ mol L⁻¹ to 5.2 × 10⁻³ mol L⁻¹ with a detection limit of 5.0 × 10⁻⁹ mol L⁻¹. Moreover, the biosensor exhibited good reproducibility and long-term stability.