Monitoring the assembly of macromolecules to design entities with novel properties can be achieved either chemically creating covalent bonds or by non-covalent connections using appropriate structural motifs. In this report， two self-associating peptides (named K3 and E3) that originate from p53 tetramerization domain were developed as tools for highly specific and non-covalent heterotetramerization of two bio-molecules. The pairing/coupling preferences of K3 and E3 were first evaluated by molecular modeling data and confirmed using circular dichroism spectroscopy， size-exclusion chromatography and biological assays. Regardless of the moieties fused to K3 and E3， these two peptides self-assembled into dimers of dimers to form bivalent heterotetrameric complexes that proved to be extremely stable inside of living cells. The benefits of the multivalency in terms of avidity， specificity and expanded functional activity were strikingly revealed when the Proliferating Cell Nuclear Antigen (PCNA)， which is essential for DNA replication， was targeted using a heterotetramer presenting both an antibody fragment against PCNA and a specific PCNA binder peptide. In vitro heterotetramerization of these two known PCNA ligands increased their binding efficiencies to PCNA up to 80-fold compared to the best homotetramer counterpart. In cellulo， the heterotetramers were able to efficiently inhibit DNA replication and to trigger cell death. Altogether， we demonstrate that these two bi-selective self-assembling peptidic domains offer a versatile non-covalent conjugation method that can be easily implemented for protein engineering.