Codelivery of diagnostic probes and therapeutic molecules often suffers from intrinsic complexity and premature leakage from or degradation of the nanocarrier. Inspired by the "Y" shape of indocyanine green (ICG), the dye is integrated in an amphiphilic lipopeptide (RNF). The hydrophilic segment is composed of arginine-rich dendritic peptides, while cyanine dyes are modified with two long carbon chains and employed as the hydrophobic moiety. They are linked through a disulfide linkage to improve the responsivity in the tumor microenvironment. After formulation with other lipopeptides at an optimized ratio, the theranostic system (RNS-2) forms lipid-based nanoparticles with slight positive zeta potential enabling efficient condensation of DNA. The RNS-2 displays glutathione responded gene release, activatable fluorescence recovery, and up to sevenfold higher in vitro transfection than Lipofectamine 2000. Compared with a Cy3 and Cy5 labeled fluorescence resonance energy transfer indicator for gene release, the "turn-on" indocyanine green analogs exhibit longer emission wavelength and better positive correlation with the dynamic processes of gene delivery. More importantly, the RNS-2 system enables efficient near infrared imaging guided gene transfer in tumor-bearing mice and thus provides more precise and accurate information on location of the cargo gene and synthesized carriers. © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.