A novel multifunctional Pt nanoparticle@PPy nanofiber intercalated structure (Pt NP@PPy NF) has been synthesized facilely in one-pot. Pt NPs, with size and facet control, were nicely assembled and embedded into the polymer nanofiber network. Polyvinylpyrrolidone (PVP) was used during the synthesis process which would assist the self-assembly of the metal nanoparticles and polymer backbones into the intercalated structure. Space-confined distribution of the Pt NPs was achieved within the large dimension PPy nanofiber network, which could enhance the interfacial electron transfer process as well as diminish the catalyst deformation. The as-formed Pt NPs have a cluster-like structure and are mainly composed of 3.5 nm primary Pt particles with (100) surface atoms. Enhanced electrocatalytic properties were shown by the Pt NP@PPy NF intercalated structure, with sufficiently high enzyme-less glucose biosensitivity and a long linear range from 1-30 mM (R = 0.9995). High electrochemical cycling stability, chloride (Cl(-)) tolerance and good selectivity are also obtained for the Pt NP@PPy NF structure, as the electrode showed no obvious response to the common interfering agents, such as ascorbic acid (AA), uric acid (UA), and 4-acetamidophenol (AP). Furthermore, the Pt NP@PPy NF showed excellent catalytic activity for the methanol oxidation reaction (MOR) and oxygen reduction reaction (ORR), which displayed sufficient CO tolerance, and higher activity compared to the commercial Pt/C catalyst. This intrinsically multifunctional Pt NP@PPy NF with well-controlled Pt facets thus could serve as an advanced electrocatalyst for biosensing and fuel cell applications, surpassing the performance of many existing materials.