degradation. [4][5][6][7] Intracellular pH is tightly regulated within a narrow range (6.8-7.2) in the cytosol through the ability of membrane proton pumps and transporters. An abnormal pH level has also been demonstrated to trigger many diseases including Alzheimer's disease, cancer, and stroke. [8][9][10][11] Hence, designing stable and reliable probes for the detection of intracellular pH changes is necessary for studying intracellular events and would be critical to understand the biological role of pHi.Semiconducting polymer nanoparticles (SPNs) have recently shown appealing performances as imaging probes [12][13][14] and therapeutic agents [15,16] owing to their high fluorescence brightness, nonblinking behavior, excellent photostability, and good biocompatibility. Owing to the amplified energy transfer ability of SPNs, some fluorescence resonance energy transfer (FRET)based SPNs probe for ratiometric detection of metal ion, [17][18][19] biomolecule, [20][21][22] as well as intracellular pH value [23,24] and temperature [25] have been reported. However, most of these systems are based on energy transfer from SPNs to small molecules, which are either susceptible to photobleaching or easy to leach from SPNs. Furthermore, direct coprecipitation of reference dye and signal indicator may pose an interference of target-responsive group on reference dye emission. Therefore, the exploration of SPNs-based ratiometric imaging probes, which combines structural as well as optical stability and consistent ratiometric emission intensities are still highly desired but remain to be revealed.Here, we propose a new approach for the preparation of ratiometric probes (PFO/PFV-DA NPs) with a previously unreported core-shell structure for intracellular pH sensing. Semiconducting polymer PFO and PFV were selected as central donor and shell acceptor respectively because both PFO and PFV exhibit signal amplification property, excellent photostability, and biocompatibility. In additional, core-shell structured ratiometric nanoprobe with all semiconducting polymer composition will reduces leaching of materials, which is ideal for intracellular sensing and in vivo imaging. Importantly, Our PFO/PFV-DA NPs have completely different structure of PFO/PFV core-shell compared to previous reports, where PFO Highly stable semiconducting polymer nanoparticles (NPs) (poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO)/ poly(fluorene-2,7-ylenevinylene-cophenylene) (PFV)-dopamine (DA) NPs) with previously unreported core-shell structure are developed for ratiometric sensing of intracellular pH values. PFO/ PFV-DA NPs comprise central polyfluorene (PFO) as donor and PFV as acceptor, in which the donor and acceptor are spatially separated into the central core and nanoparticle shell. Specifically, thick PFV shells can not only significantly minimize the quenching interference of dopamine on the emission of standard reference (PFO), but are also able to maximize its accessibility to pH-sensitive dopamine and lead to sensitive response to pH changes. The resul...