Fluorescence-guided surgery (FGS) is an emerging and highly promising surgical technique in clinic. Owing to its real-time and visual characteristics, it assists in achieving clear pictures on lesion site, tumor boundary and degree of metastasis, which will definitely improve surgery accuracy and minimize cancer recurrence as much as possible. Herein, we report a near-infrared fluorescent bioprobe, YK80, which utilizes a modified heptamethine cyanine dye as the fluorophore and a self-assembling peptide targeting Ephrin receptor A2 (EphA2) proteins as the ligand. The design strategy and the synthetic route to YK80 are described, and then optical properties, pharmacokinetics, binding affinity between YK80 and the protein are further investigated. YK80 shows high affinity (K