A novel dual-emission carbon dots (CDs) based fluorescence strategy was developed for the ratiometric detection of Fe3+ and pyrophosphate (PPi) in biological samples. The fluorescence of blue CDs (b-CDs) at 450 nm was remarkably quenched by Fe3+ due to photoinduced electron transfer (PET), whereas the fluorescence peak at 550 nm of yellow CDs was unaffected and could serve as an internal standard. In addition, the intense coordination between PPi and Fe3+ separated Fe3+ from the b-CD surface and blocked the PET process, thus effectively protecting the fluorescence of the b-CDs. The ratiometric signal changes enabled sensitive and robust Fe3+ and PPi sensing. Good linearities for the detection of Fe3+ and PPi were obtained in the ranges of 1.0 − 60.0 μM and 0.1 − 120.0 μM with detection limits of 0.28 μM and 0.032 μM, respectively. Furthermore, the developed strategy was successfully used to determine Fe3+ and PPi in biological samples. The proposed probe may offer a simple, sensitive, environmentally friendly, and promising platform for biological detection applications.