Site-specific and fragment-selective internal labeling of double-stranded DNA (dsDNA) remains a fundamental challenge, as most existing strategies rely on thermal denaturation, predefined sequence motifs, or enzymatic incorporation or are limited to end-labeling. Concurrently, conventional peptide nucleic acids (PNAs) exhibit poor invasion efficiency in mixed-sequence and GC-rich dsDNA due to insufficient binding energy and limited conformational preorganization. Here, we report a chimeric γPNA–peptide invasion probe that enables programmable, site-specific invasion and internal fluorescence labeling of dsDNA under mild and enzyme-free conditions. Partial γ-backbone modification of the PNA and conjugation with an SV40 nuclear localization signal enhances helical preorganization and binding interactions, overcoming thermodynamic barriers associated with GC-rich targets. When combined with T7 endonuclease I (T7EI), our invasion probe forms a PAM-free, programmable toolbox for fragment-specific labeling and site-specific cleavage of dsDNA, surpassing conventional end-labeling strategies. The invasion probe directs T7EI cleavage at the invasion site, generating fragments that are stably associated with the probe. Importantly, this inherently universal platform can be readily extended to new targets by changing the probe sequence. Altogether, this work establishes a versatile and programmable toolbox for fragment-specific dsDNA labeling and cleavage with minimal sequence constraints, opening new opportunities in molecular diagnostics, gene regulation, and DNA nanotechnology.