A superior NADH sensing platform was constructed based on porous PtAg nanoshells supported on reduced graphene oxide (PtAg/rGO) in the absence of any enzymes and redox mediators. The PtAg/rGO composite was prepared via one-step reduction combined with galvanic replacement reaction. The as-made PtAg/rGO assembles multiple structural advantages of coherent conductive matrix, rich electroactive sites, and high specific surface area, accompanied by the unique alloying effect. The PtAg/rGO possesses adequate active reaction sites and fluent electron transport pathway towards the electrocatalytic NADH oxidation, thus presenting significantly increased oxidation current and negative shift of 330 mV in applied potential relative to the bare GCE. By virtues of the outstanding electrocatalytic activity, PtAg/rGO exhibits effective amperometric detection of NADH at 0.15 V within a wide linear concentration range of 2–2378 μM, a high sensitivity of 92.62 μA mM−1 cm−2, low detection limit of 0.2 μM, and long-term detection over 2500 s. Moreover, the as-constructed biosensors can achieve accurate NADH detection in human serum samples, indicating its promising application feasibility in fundamental and clinic research. Graphical AbstractPorous PtAg alloy nanoshells supported on reduced graphene oxide (PtAg/rGO) was prepared via a facile one-step reduction and spontaneous replacement reaction strategy. A sensitive and highly stable electrochemical biosensor based on PtAg/rGO is constructed for the quantitative detection of NADH at low applied potential.