Cyclic peptides are drawing wide attention as potential medium-sized modulators of biomolecular interactions with large binding surfaces. Simple but effective peptide cyclization methods are needed to construct cyclic peptide libraries by both peptide and nonpeptide chemists. Herein, we report a highly chemoselective and operation-simple method directly cyclizing unprotected peptides, in which ortho-phthalaldehyde (OPA) is found to react with the lysine/N-terminus and cysteine within one unprotected peptide sequence effectively to form the isoindole-bridged cyclic peptides. This reaction is carried out in the aqueous buffer and features tolerance of diverse functionalities, rapid and clean transformation, and operational simplicity. In addition, OPA peptide cyclization can also be combined with native chemical ligation-mediated cyclization to generate bicyclic peptides. Furthermore, the OPA peptide cyclization product can further react with the N-maleimide moiety in a one-pot manner to introduce additional functional motifs, like a fluorophore probe, biomolecules (e.g., glycan, peptide, or DNA). This OPA-cyclization method extends the toolbox for integrating postcyclization modification and bioconjugation into peptide cyclization with an all-in-one manner strategy.