Cell penetrating peptides (CPPs) are very useful tools for delivery of DNA molecules into living cells without damaging the cell membranes. However, covalent conjugation of DNAs to CPPs is technically difficult, and the reactions between DNA and target nucleases are also liable to be affected by the cationic CPP molecules. In this work, we demonstrate that the electrostatic interactions between CPPs and single-stranded DNA (ssDNA) were stronger than those between CPP and double-stranded DNA (dsDNA). Taking advantage of this property, we developed an ssDNA protected CPP-DNA fluorescent probe which allowed for noninvasive and efficient cellular uptake and rapid imaging of target nucleases in living cells. The probe is highly sensitive and selective. This work represents the first example of using CPP-DNA conjugate to deliver DNA fluorescent probes for in situ imaging of nucleases within cells. The developed approach also holds great potential for the cellular delivery of other nucleic acid molecules for diagnosis or therapeutics purposes.