Nasopharyngeal carcinoma (NPC) is one of the most common tumors in South China and Southeast Asia and is thought to be associated with Epstein-Barr virus (EBV) infection. Downregulation of latent membrane protein 1 (LMP1) encoded by EBV can reduce the expression of NF-κB and PI3K, induce apoptosis, and inhibit the growth of EBV-related NPC. For targeted cleavage of the Lmp1 oncogene via the CRISPR/Cas9 gene editing system, a post cross-linked ROS-responsive poly(β-amino ester) (PBAE) polymeric vector was developed for the delivery of CRISPR/Cas9 plasmids both in vitro and in vivo. After composition optimization, the resultant polymer-plasmid polyplex nanoparticles (NPs) showed a diameter of ∼230 nm and a zeta potential of 22.3 mV with good stability. Compared with the non-cross-linked system, the cross-linked NPs exhibited efficient and quick cell uptake, higher transfection efficiency in EBV-positive C666-1 cells (53.5% vs. 40.6%), more efficient gene editing ability against the Mucin2 model gene (Muc2) (17.9% vs. 15.4%) and Lmp1 (8.5% vs. 5.6%), and lower intracellular reactive oxygen species (ROS) levels. The NPs achieved good tumor penetration and tumor growth inhibition in the C666-1 xenograft tumor model via Lmp1 cleavage, indicating their potential for gene therapy of EBV-related NPC.