<p>The functional design of carbon dots (CDs) is the key to enabling their practical biological applications. In this study, mitochondria-targeted carbon dots (M-CDs) with a large Stokes shift were successfully synthesized using Rhodamine B, o-phenylenediamine and pentyltriphenylphosphonium bromide as precursors through a one-step solvothermal method. Stable fluorescence performance of M-CDs was demonstrated in environments containing various ions (except Fe³⁺), amino acids, as well as under different pH, polarity, and viscosity conditions, guaranteeing their reliable application in complex biological environments. M-CDs exhibited highly efficient and specific mitochondrial targeting through a “synergistic electrostatic-hydrophobic targeting mechanism” as evidenced by a co-localization coefficient of 0.91 with a commercial mitochondrial probe. Furthermore, M-CDs were successfully employed to monitor mitochondrial morphological changes in real-time during hydrogen peroxide-induced oxidative stress and CCCP-triggered mitophagy. In addition, it was found that M-CDs exhibited unexpected specific fluorescence response to Fe³⁺, with a static quenching mechanism and a detection limit of 0.65 µM. Finally, M-CDs served as a sensitive indicator of intracellular iron overload, offering key evidence for the occurrence of ferroptosis. This study not only developed a highly stable multifunctional fluorescent carbon dot but also revealed the synergistic effect of the lipophilicity of long-chain alkyl groups and electrostatic guidance of TPP cations in enhancing organelle targeting, providing new ideas for the design of organelle imaging probes for complex biological environments.</p> Graphical Abstract <p></p>