Copper ions are regarded as a double-edged sword since it plays a crucial role in biological functions but can also induce various severe diseases at imbalanced levels. However, conventional methods for quantifying copper ion (Cu2+) levels often encounter complicated preparation procedures and interference from other substances. This study describes the one-pot microwave synthesis of sulfur-doped carbon dot (S_HICA) as a highly selective and sensitive fluorescent probe for Cu2+ detection in biological systems. S_HICA demonstrates improved fluorescence properties such as increased intensity and a red-shifted emission wavelength compared to carbon dots without sulfur (HICA), and S_HICA exhibits outstanding selectivity for Cu2+ through a fluorescence quenching effect. In vitro and ex vivo experiments are performed to validate the fluorescence selectivity of S_HICA, revealing that S_HICA has remarkable biocompatibility, fluorescence stability, and tolerance to varying pH levels. S_HICA presents significant potential as a fluorescent probe for detecting Cu2+ with elevated sensitivity and selectivity, as well as for the advancement of enhanced diagnostic tools that can monitor copper-related disorders in real time.