Intracellular pH plays a significant role in all cell activities. Due to their precise imaging capabilities, fluorescent probes have attracted much attention for the investigation of pH regulated processes. Detecting intracellular pH values with high throughput is critical for cell research and applications. In this work, hybrid semiconducting polymer dots (Pdots) were developed and characterized and were applied for cell imaging and exclusive ratiometric sensing of intracellular pH values. The reported Pdots were prepared by blending a synthesized block polymer (POMF) and a semiconducting polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEHPPV) to construct a fluorescence resonance energy transfer system for ratiometric sensing. Pdots showed many advantages, including high brightness, excellent photostability and biocompatibility, giving the pH probe high sensitivity and good stability. Our results proved the capability of POMF–MEHPPV Pdots for the detection of pH in living cells.