Three phosphono-containing multidentate ligands were employed to synthesize quinquedentate binuclear copper complexes, [Cu2L2] (1–3) (H2L1 = diethyl(propane-1,3-diylbis(azanediyl))bis((2-hydroxyphenyl)methylene)bis(hydrogen phosphonate), H2L2 = diethyl(ethane-1,2-diylbis(azanediyl))bis((2-hydroxyphenyl)methylene)bis(hydrogen phosphonate), H2L3 = diethyl(hexane-1,6-diylbis(azanediyl))bis((2-hydroxyphenyl)methylene)bis(hydrogen phosphonate)), which were characterized by elemental analysis, IR, X-ray diffraction analysis, electrospray ionization mass spectra. Complexes 1 and 2 crystallized in the triclinic system with space group P
. The speciation of the Cu–H2L1 system in aqueous solution was investigated by potentiometric pH titrations. The three dicopper complexes exhibited potent and almost the same inhibitory effects against protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TCPTP) with IC50 of 0.16–0.24 μM, about 10-fold stronger inhibition than against Src homology phosphatase 1 (SHP-1), 30-fold than against Src homology phosphatase 2 (SHP-2) and more than 100-fold than against megakaryocyte protein-tyrosine phosphatase 2 (PTP-MEG2). Fluorescence titrations revealed complex 1 bond to the five PTPs with molar ratio of 1 : 1 and binding constants of 1.62 × 106, 3.09 × 106, 1.95 × 105, 2.24 × 105, 1.55 × 104 M−1 for PTP1B, TCPTP, SHP-1, SHP-2 and PTP-MEG2, respectively, consistent with the inhibitory abilities from IC50 and Ki values. Also, the three copper complexes could inhibit phosphatase activity of cell extracts from C6 rat glioma cells. The results suggested the structures of copper complexes influence selectivity over different PTPs.