Combination chemotherapy in clinical practice has been generally accepted as a feasible strategy for overcoming multidrug resistance (MDR). Here, we designed and successfully prepared a co-delivery system named S-D1@L-D2 NPs, where denoted some smaller nanoparticles (NPs) carrying a drug doxorubicin (DOX) were loaded into a larger NP containing another drug (vincristine [VCR]) via water-in-oil-in-water double-emulsion solvent diffusion-evaporation method. Chitosan-alginate nanoparticles carrying DOX (CS-ALG-DOX NPs) with a smaller diameter of about 20 nm formed S-D1 NPs; vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying VCR (TPGS-PLGA-VCR NPs) with a larger diameter of about 200 nm constituted L-D2 NPs. Some CS-ALG-DOX NPs loaded into TPGS-PLGA-VCR NPs formed CS-ALG-DOX@TPGS-PLGA-VCR NPs. Under the acidic environment of cytosol and endosome or lysosome in MDR cell, CS-ALG-DOX@TPGS-PLGA-VCR NPs released VCR and CS-ALG-DOX NPs. VCR could arrest cell cycles at metaphase by inhibiting microtubule polymerization in the cytoplasm. After CS-ALG-DOX NPs escaped from endosome, they entered the nucleus through the nuclear pore and released DOX in the intra-nuclear alkaline environment, which interacted with DNA to stop the replication of MDR cells. These results indicated that S-D1@L-D2 NPs was a co-delivery system of intracellular precision release loaded drugs with pH-sensitive characteristics. S-D1@L-D2 NPs could obviously enhance the in vitro cytotoxicity and the in vivo anticancer efficiency of co-delivery drugs, while reducing their adverse effects. Overall, S-D1@L-D2 NPs can be considered an innovative platform for the co-delivery drugs of clinical combination chemotherapy for the treatment of MDR tumor.