Developing antibiotics-independent antibacterial agents is of great importance since antibiotic therapy faces great challenges from drug resistance. Graphene oxide (GO) is a promising agent due to its natural antibacterial mechanisms, such as sharp edge-mediated cutting effect. However, the antibacterial activity of GO is limited by its negative charge and low photothermal effect. Herein, the amino-functionalized GO nanosheets (AGO) with unique three-in-one properties were synthesized. Three essential properties (positive charge, strong photothermal effect, and natural cutting effect) were integrated into AGO. The positive charge (30 mV) rendered AGO a strong interaction force with model pathogen Streptococcus mutans (330 nN). The natural cutting effect of 100 μg·mL-1 AGO caused 27% loss of bacterial viability after incubation for 30 min. Most importantly, upon the near-infrared irradiation for just 5 min, the three-in-one properties of AGO caused 98% viability loss. In conclusion, the short irradiation period and the tunable antibacterial activity confer the three-in-one AGO a great potential for clinical use.