Herein, we disclose a Rh(III)-catalyzed C-H activation/annulation reaction for the derivatization of Lys-based peptides, in situ affording diverse peptide-isoquinolone conjugates. This approach features racemization-free conditions, high atom- and step-economy, excellent chemo- and site-selectivity, and broad scope including substrates bearing unprotected Trp and Tyr, free Ser and Gln, and Met residues. The peptide-isoquinolone conjugates also display good fluorescent properties with maximum emission wavelengths up to 460 nm. Importantly, preliminary antifungal activity studies indicate that peptide-isoquinolone conjugates show potential activities toward crop and forest pathogenic fungi, in which the peptide-isoquinolone conjugate bearing unprotected Tyr residue exhibits much better antifungal activities toward B. cinerea Pers. and C. chrysosperma than the positive control.