Core‐shell tecto dendrimers (CSTDs) with excellent physicochemical properties and good tumor penetration and gene transfection efficiency have been demonstrated to have the potential to replace high‐generation dendrimers in biomedical applications. However, their characterization and related biological properties of CSTDs for enhanced tumor penetration and gene delivery still lack in‐depth investigation. Herein, we attempted to design three types of dual‐responsive CSTDs for thorough physicochemical characterization and investigation of their tumor penetration and gene delivery efficiency. Three types of CSTDs were prepared through phenylborate ester bonds of phenylboronic acid (PBA)‐decorated generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers as cores and monose (galactose, glucose or mannose)‐conjugated G3 PAMAM dendrimers as shells and thoroughly characterized via NMR and other techniques. We show that the produced CSTDs displayed strong correlation signals between the PBA and monose protons and similar hydrodynamic diameters, and possess dual reactive oxygen species‐ and pH‐responsivenesses. The dual‐responsive CSTDs were proven to have structure‐dependent tumor penetration property and gene delivery efficiency in terms of small interference RNA for gene silencing and plasmid DNA for gene editing, thus revealing a great potential for different biomedical applications.This article is protected by copyright. All rights reserved