Nitrogen (N) and sulfur (S) heteroatoms co-doping has been confirmed to be an effective strategy to significantly promote the electrocatalytic activity of graphene. However, developing N, S co-doped graphene (NS–G) with a high doping level at low cost remains a great challenge. Meanwhile, NS–G has rarely been investigated for electrochemical sensing. Here, a one-step and cost-effective microwave-assisted solvothermal method was developed to synthesize NS–G having a high N, S-doping level (N: 6.8at.%, S: 2.1at.%). When employed as an electrocatalyst for H2O2 reduction, NS–G exhibited a higher electrocatalytic activity than its undoped or mono-doped counterparts due to the synergistic effect of N, S co-doping. Consequently, the sensors based on NS–G displayed good sensing performances for sensitive detection of H2O2 (0.1–16.6mM in linear range, 0.2μM in detection limit) and glucose (0.1–12.6mM, 0.5μM), demonstrating the potential application of NS–G for electrochemical sensing.