Susceptibility point-of-care testing (POCT) and bacterial inactivation are crucial for food safety and clinical diagnosis since bacteria pose a severe hazard to public health and healthcare. To enable photothermal determination and hyperthermal inactivation of bacteria, vancomycin-modified near-infrared (NIR)-responsive copper selenide nanoparticles (CuSe@Van NPs) with excellent photothermal conversion are synthesized here. CuSe@Van NPs can be trapped on the surface of Gram-positive bacteria such as Staphylococcus aureus (S. aureus), forming a CuSe@Van NPs/S. aureus complex. Suspension (CuSe@Van NPs) and sediment (CuSe@Van NPs/S. aureus) can convert the NIR laser energy into a thermal signal that can be detected by the thermal camera at concentrations as low as 6.0 CUF mL. Meanwhile, S. aureus can be effectively inactivated by localized temperature rises during temperature-based signal detection. The antibacterial efficiency can reach as high as 99.53%. The proposed strategy not only provides a sensitive POCT strategy for bacterial determination but also implements efficient inactivation of bacteria simultaneously.