Monitoring gene delivery has significant benefits in gene therapy. Herein, we report a nanoquencher system by doping a FRET pair during nucleic acid-assisted cell penetrating poly(disulfide) (CPD) formation. Our results show that this strategy not only produces an efficient gene delivery polymer with minimal endolysosomal trapping, but also enables monitoring the release of the gene from the vehicle in live cells. This study further expanded the application of CPDs as promising tools in gene delivery.