The multidomain adaptor protein syntenin is known to mediate cancer cell metastasis and invasion through its tandem PDZ1 and PDZ2 domains， leading to the postulation that the PDZ tandem may serve as a potential drug target for cancer treatment. Here we report the development of high-affinity peptide blockers to target the syntenin tandem PDZ domain， and elucidate that blocking syntenin correlates with the inhibition of cell migration and spreading. Two strategies are employed to derive high-affinity blockers from the low-affinity natural binding peptides: first， dimerization of the C termini of natural syntenin-binding peptides confers dimer peptides with much higher affinity than the monomers; second， unnatural amino acid substitution at P-1 and P-2 positions of the PDZ-binding sequence increases the binding affinity. Through several rounds of optimization， we discovered a dimeric peptide that binds tightly to syntenin tandem PDZ domain， with a dissociation constant of 0.21 μM based on fluorescence polarization measurement. The peptide dimer inhibits the migration and invasion of syntenin high-expression human cancer cells through attenuating the ERK phosphorylation of the MAPK kinase pathway. This work showcases an effective strategy to derive high-affinity blocker of multidomain adaptor proteins， which resulted in a syntenin-targeted antagonist with potential pharmaceutical values for the treatment of syntenin over-expressing cancers.