A strategy for one‐pot and large‐scale synthesis of functionalized core–shell nanowires (NWs) to high‐efficiently construct single nanowire electrodes is proposed. Based on the polymerization reaction between 3,4‐ethylenedioxythiophene (EDOT) and noble metal cations, manifold noble metal nanoparticles‐polyEDOT (PEDOT) nanocomposites can be uniformly modified on the surface of any nonconductive NWs. This provides a facile and versatile approach to produce massive number of core–shell NWs with excellent conductivity, adjustable size, and well‐designed properties. Nanoelectrodes manufactured with such core–shell NWs exhibit excellent electrochemical performance and mechanical stability as well as favorable antifouling properties, which are demonstrated by in situ intracellular monitoring of biological molecules (nitric oxide) and unraveling its relevant unclear signaling pathway inside single living cells. Versatile one‐pot synthesis of functionalized core–shell nanowires breaks through the limitation of nanoelectrode materials to facilely construct high‐performance single nanowire electrodes. Concurrently with excellent electrochemical, mechanical, and antifouling properties, the nanowire electrodes show great superiority in real‐time monitoring of biological molecules and unraveling the relevant signaling pathway inside single living cells.