Abstract “Turn-on” fluorescent materials in the absence of metal and organic solvent are highly desirable as bioprobes to selectively and sensitively detect pyrophosphate (PPi) anion. In this work, we embedded tetraphenylethene (TPE) inside the relatively rigid hyperbranched poly(amido amine) (HPA) to afford highly water soluble TPE-HPA materials, where the TPE fluorescence was pre-quenched in water by the hyperbranched structure of HPA without participation of any metal or organic solvents. The obtained TPE-HPA could be directly used as fluorescence “turn-on” bioprobe under physiological conditions to selectively detect PPi. There was a good linearity between the photoluminescence enhancement and the PPi concentration in the range of 0–1.0 μM with a limit of detection (LOD) of 6.5 nM. Compared with the other reported AIE probes for PPi detection, TPE-HPA with the optimal TPE content showed the best comprehensive performance, that is metal-free, organic-solvent free and nanomolar level LOD. The extreme selectivity and sensitivity towards PPi allowed TPE-HPA to accurately detect the PPi concentration in human urine.