A tumor-responsive nanoprobe based on a conjugated polyelectrolyte and gold nanoparticle (AuNP) hybrid was designed to response to the low pH extracellular microenvironment in tumor with light-up fluorescence. AuNPs with positive surface charges were prepared by direct reducing Au salt with sodium borohydride and stabilized by cystamine. A pH triggered charge-reversible polymer and a water-soluble cationic conjugated polyelectrolyte (CPE) were sequentially deposited onto the AuNP surface through electrostatic interaction. The obtained hybrid probe is monodispersed with an average diameter of 68.3 nm by dynamic light scattering measurement. In physiological conditions (pH ≈ 7.4), the hybrid probe is almost non-fluorescent due to the super-quenching of CPE by AuNPs via energy/charge transfer and efficient exciton migration along the polymer backbone. When exposed to acidic extracellular microenvironments in tumor (pHe ≈ 6.5), the acid-labile amides hydrolyze into primary amines. The generated amine groups result in strong electrostatic repulsion between CPE and AuNPs, leading to recovered probe fluorescence. The fluorescence turn-on is further utilized for tumor extracellular acidic microenvironment imaging. In addition, under in vivo conditions, the nanosized hybrid probe exhibits specific accumulation in tumor tissue with light-up fluorescence, which provides new opportunities for easy tumor imaging and identification.