Protein detection in complicated biological samples requires robust design and a rigorous rinsing process. Many recently developed artificial targeting probes, however, often do not possess antibody-like binding strength that enables them to endure harsh biosensing conditions, and the classic 2-to-1 sandwich binding pattern is unavailable for many targets, often necessitating a complicated indirect signal conversion mechanism. Here, an attempt is made to provide innovative "covalent" solutions to such problems by employing peptide reactivity to form a covalent and robust biosensing structure upon target binding. Both the cross-coupling and cleavage of peptide chains are employed to achieve the classic 2-to-1 binding when only one targeting probe is available. Specifically, a targeting probe against the protein and a signaling probe are coimmobilized onto the sensing interface. The ratio between the two probes and their surface density is modulated so that direct cross-linking between the two immobilized probes is suppressed by the average distance between two such probes. Upon protein binding, the protein molecule may bridge that gap by itself. The signaling probe can carry any motif of signal amplification. And here a Cu-ion-complexed motif, which can exhibit peroxidase-like activity upon electrochemical agitation, is employed multipurposely as the catalyst for cross-linking, cleavage, and signal amplification. Three nonhomologous target proteins can be sensitively quantified in serum-spiked samples and clinical sample detection of one of them is also successful; these results suggest the potential of the proposed method in future clinical applications.