The immune checkpoint blockade (ICB) of programmed death ligand-1 (PD-L1) or programmed death-1 (PD-1) has gained promising performances for triggering durable response and manageable toxicity of the host's antitumor immunity. However, ICB therapy is still unsatisfactory considering the restrained tumor-infiltrating lymphocytes (TILs), poor accumulation and penetration of antibodies and immunosuppressive tumor microenvironment (TME). Herein, we developed a two-step sequential delivery strategy for potent immune cocktail therapy to amplify anti-PD-L1 immunotherapy. Quercetin (Que) was coated by bovine serum albumin (BSA) to form nanoparticles (QB NPs) for remodeling tumor microenvironment (TME) to increase the penetration of the subsequent nanoparticles (NPs) in tumor issues and promote T cell infiltration. Consequently, the nucleotide drug carrier, PF180, coated with pH-sensitive polyethyleneglycol (PEG) was fabricated to co-deliver unmethylated cytosine-phosphate-guanine (CpG) and plasmid DNA encoding small hairpin RNA of PD-L1 (pshPD-L1). Such nanoparticles (NPs) with high efficiency and low toxicity could significantly induce dendritic cell maturation and PD-L1 downregulation on tumor cell surface. This sequential delivery strategy effectively ameliorated the harmful extracellular matrix (ECM) via reducing the activity of tumor-associated fibroblasts (TAF). Together with the reduced expression of collagen and ɑ-SMA in tumor tissue, the silenced PD-L1 gene can strongly inhibit tumor growth. The infiltration of immune cells was also enhanced, favoring the reversal of the immunosuppressive tumor microenvironment. This combination treatment provided a feasible strategy to improve the antitumor efficacy of anti-PD-L1 cancer therapy.