A series of fluorescent multiblock PEG-polyurethane copolymers with free carboxyl groups were synthesized by one-step method and their chemical structure and properties were investigated. The multiblock PEG-polyurethane copolymers were composed of poly(ethylene glycol) (PEG), 1,6-hexamethylene diisocyanate, 2,2-bis (hydroxymethyl) propionic acid and a fluorescent dye erythrosine B (EB). The resulting polymers were examined by 1HNMR, FT-IR, gel permeation chromatography (GPC), ultraviolet visible spectroscopy (UV-vis), fluorescent spectroscopy. As a result, the UV-vis spectra showed that EB can be successfully conjugated into PEG-polyurethane during the one-step reaction. And the UV absorption peaks of samples were blue-shift and strengthened with decreasing of pH values, indicating that PEG-polyurethane-EB can be a pH-dependent fluorescent probe in a broad pH range. We expect that more applications would be possible for the fluorescent PEG-polyurethane with free carboxyl groups owing to its free functional groups and simultaneously imaging along with pH values.