Heparan sulphate proteoglycans (HSPGs) are cell surface and extracellular matrix proteoglycan family members, playing a key role in diverse biological activities including signal transduction or endocytosis. To date, many HSPG ligands have been identified, including growth factors interacting via sulfated domains of heparan sulphate (HS) chains. Due to significant overexpression of HSPGs in various cancers, discovering novel biomolecules capable of HSPGs recognition, which may act as HSPGs biosensors or drug carriers targeting HSPGs, is essential. Fibroblast growth factors (FGFs), mainly paracrine FGFs, are natural ligands of membrane-bound HSPGs, forming ternary signaling complex with HSPGs and fibroblast growth factor receptors (FGFRs). Here we employed the natural capability of FGF1 to bind HSPGs to develop HSPGs-specific proteinaceous probe. We identified three point mutations within FGF1 that elevate its affinity for heparin-S116R, S17K and L72R, named B, C and D respectively. Together with substitutions increasing FGF1 stability and abolishing FGF1 binding to FGFRs, we have generated eight HSPGs-specific variants. Among tested mutants, FGF1BCD exhibits the highest affinity for heparin and HS/HSPGs, showing over 20-fold increase in affinity for glypican-4 and requiring 0.23 M higher salt concentration for elution from heparin column compared to the initial FGF1 molecule. Finally, we demonstrated FGF1BCD potential to act as a molecular sensor of HSPGs level in cancer cell lines overproducing HSPGs, implicating that FGF1BCD can be used as HSPGs biosensor or as a drug delivery carrier in protein-drug conjugates (PDC) targeting HSPGs.