A facile route to prepare fluorescent carbon dots was developed by hydrothermal treatment of sodium gluconate. We further reported the incoporation of doxorubicin molecules into the prepared carbon dots/phospholipid hybrids that was synthesized by a self-assembly of a phosphatidylcholine monolayer on the surface of carbon dots. And we demonstrated the fluorescence resonance energy transfer (FRET) process between the fluorescence carbon dots and doxorubicin in the nanohybrid. The phospholipase C can catalyze the hydrolysis of phospholipid, which disrupts the carbon dots-phospholipid-doxorubicin complex. Herein, the developed FRET system was utilized as a ratiometric fluorescence sensor for phospholipase C detection in aqueous and bioimaging in breast cancer cell.