To increase the efficacy of small molecule chemotherapeutic drug (SMCD) and reduce its toxic and side effects, we selected two model drugs doxorubicin (DOX) and chloroquine (CQ). DOX is a SMCD and CQis a chemosensitizer with autophagy inhibition. Poly(lactic-co-glycolic acid) (PLGA) and alpha-tocopherol polyethylene glycol 1000 succinate were chosen as delivery carriers to design and prepare a novel type of drug co-delivery single-nanoparticles by emulsification-solvent volatilisation, named NPDOX+CQ. The physicochemical properties of NPDOX+CQ were characterised. Then A549 cells and A549/Taxol cells were used for the in vitro anti-cancer effect study. At the same time, cellular uptake, intracellular migration and anti-cancer mechanism of nanoparticles were studied. The NPs showed a uniform spherical shape with good dispersibility, and both drugs had good encapsulation efficiency and loading capacity. In all formulations, NPDOX+CQ showed the highest in vitro cytotoxicity. The results showed that NPs could protect drugs from being recognised and excluded by P-glycoprotein (P-gp). Moreover, the results of the mechanistic study demonstrated that NPs were transported by autophagy process after being taken up by the cells. Therefore, during the migration of NPDOX+CQ, CQ could exert its efficacy and block autophagy so that DOX would not be hit by autophagy. Western Blot results showed that NPDOX+CQ had the best inhibition effect of autophagy. It can be concluded that the system can prevent the drug from being recognised and excluded by P-gp, and CQ blocks the process of autophagy so that the DOX is protected and more distributed to the nucleus of multidrug resistance (MDR) cell. The NPDOX+CQ constructed in this study provides a feasible strategy for reversing MDR in tumour cells.