Heparan sulfate (HS) is a critical component of the extracellular matrix, involved in various biological processes. We synthesized three fluorescent probes (1-3) consisting of Arg-rich peptides as HS receptors and a fluorophore that exhibits red-shifted emissions when it aggregates. All three probes displayed ratiometric responses to HS and heparin in aqueous solutions. However, only probe 3 exhibited a ratiometric response to HS in aqueous solutions at physiological pH as well as HS proteoglycan on live cell. The peptide-based probe 3 demonstrated excellent sensing properties, including high biocompatibility, water solubility, visible light excitation, large Stokes shift for ratiometric detection, and high selectivity and sensitivity for HS (LOD: 720 pM). Binding mode studies revealed the crucial role of charge interactions between probe 3 and negatively charged HS sugar units. Upon binding, the fluorophore parts of the probes overlapped, resulting in green and red emission changes via restriction of the intramolecular rotation of the fluorophore moiety. Probe 3 effectively quantified the reduction of HS proteoglycan levels in live cells by inhibiting HS sulfation using siRNA and an inhibitor. It also detected decreased HS levels in cells treated with doxorubicin and irradiation, which was consistent results with those obtained from the western blot and immunofluorescence assays.  Our study presents the first ratiometric fluorescent probe for the quantitative detection of HS levels in aqueous solutions and live cells. The unique properties of peptide-based probe make it a valuable tool for studying HS biology and potentially for diagnostic applications in various biological systems.