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 (Cu_2−XSe@Van NPs) with excellent photothermal conversion are synthesized here. Cu_2−XSe@Van NPs can be trapped on the surface of Gram-positive bacteria such as Staphylococcus aureus (S. aureus), forming a Cu_2−XSe@Van NPs/S. aureus complex. Suspension (Cu_2−XSe@Van NPs) and sediment (Cu_2−XSe@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⁻¹. 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.