The bis(pyrene) conjugated pH-sensitive polymers () were synthesized and self-assembled into nanoparticles through hydrophobic interactions. The Nile red (NR) loaded nanoparticles showed red emission due to the FRET effect. The nanoparticles entered cells via endocytosis, and the microenvironmental pH in the endocytosis process was monitored in situ by the simultaneous dual-wavelength fluorescence changes.