The development of novel single-atom catalysts is important for highly efficient electrochemical catalysis and sensing. In this work, a novel Pt single atoms (SAs) supported on Ni₆Co₁ layered double hydroxides/nitrogen-doped graphene (Pt₁/Ni₆Co₁LDHs/NG) was constructed for electrochemical enzyme-free catalysis and sensing towards glucose. The loading of Pt single atoms increases with doping of Co atoms that generate more anchoring sites for Pt SAs. The resulting Pt₁/Ni₆Co₁LDHs/NG exhibits low oxidative potential of 0.440 V with high sensitivity of 273.78 μA·mM⁻¹·cm⁻² toward glucose, which are 85 mV lower and 15 times higher than those of Ni(OH)₂, respectively. Pt₁/Ni₆Co₁LDHs/NG also shows excellent selectivity and great stability during 5-week testing. Theoretical and experimental results show that the boosted performance of Pt₁/Ni₆Co₁LDHs/NG originates from its stronger binding energy with glucose and the synergistic effect of Pt SAs, Co doping, and NG. This work provides a general strategy of designing highly active SACs for extending their application in electrochemical sensing.