Nanoliposomes were first used to encapsulate two fluorophores, fluorescein and cresyl violet, to construct a FRET-based ratiometric fluorescent probe, for sensing intracellular pH values. The morphology, stability, interference factors, biocompatibility, and performance were investigated in detail. The fluorophores-encapsulated nanoliposomes (FEN) probe can be uptaken by cells within 30 min, which is faster than any other nanosensor, and cell viability was not significantly changed upon treatment with the FEN probe for 24 h, showing the low cytotoxicity and good biocompatibility of the probe. Quantitative determinations of intracellular pH of MCF-7 cells and the pH fluctuations associated with inflammation have been successfully performed with our probe. This work demonstrated that nanoliposomes could be served as an emerging approach for constructing useful fluorescent nanosensors with low toxicity.