Herein we pay attention to the design, synthesis and sensor activity of a novel biocompatible perylene-3,4,9,10-tetracarboxylic diimide (PDI) pH-probe in water. The synthesized compound shows selective fluorescence signalling properties as a function of pH (pKa value of 6.35 ± 0.02) which makes the probe suitable for pH determination in the physiological range. Thus prepared water soluble fluorescent system demonstrate low cytotoxicity to L929 cell lines from 330 μM to 1.3 μM and cell permeability in the lower concentration range. That findings show the high potential of the newly prepared PDI probe for monitoring of pH variations in bio-samples. Display omitted •Fluorescent water-soluble perylene diimide pH probe is synthesized.•Probe is configured on the “fluorophore-spacer-receptor” model.•Probe operates via photoinduced electron transfer.•Probe shows low cytotoxicity towards L929 cell line.•Nano sized probe is cell-permeable at 0.001 mg/mL concentration.