Abstract: Objective: To construct the nano-drug delivery system (GA/CUR/HCPT-NPs) of glycyrrhizic acid (GA), curcumin (CUR) and hydroxycamptocampin (HCPT) with amphiphilic drug molecules GA, hydrophobic drug CUR and HCPT as materials by self-assembly, and to investigate its preparation technology as well as to evaluate its quality. Methods: GA/CUR/HCPT-NPs was prepared by the combination of anti-solvent precipitation method and dialysis method, and the preparation process was optimized by response surface methodology (Box-Behnken). Finally, the GA/CUR/HCPT-NPs with suitable size, stable charge and high drug loading was obtained, then the optimized GA/CUR/HCPT-NPs was characterized by H nuclear magnetic resonance spectrometer (¹HNMR), differential scanning calorimetry (DSC), high-resolution X-ray diffractometer (XRD), Fourier transform infrared (FTIR) as well as ultraviolet-uisible spectropho (UV), and it's micromorphology was observed. The contents of GA, CUR and HCPT in GA/CUR/HCPT-NPs were determined. Results: The optimal prescription was 30 mL deionized water, dimethyl sulfoxide (DMSO) was dropped into the deionized water within 1 min, the preparation temperature was 40℃, and the dosage of GA, CUR and HCPT was determined to be 6.00 mg, 10.03 mg and 5.01 mg, respectively. The particle size of GA/CUR/HCPT-NPs was (146.37±0.15) nm, with a high stable charge of (-34.43±0.77) mV, and the polydispersity index (PDI) was 0.157±0.01.GA/CUR/HCPT-NPs was spherical and uniformly distributed under transmission electron microscopy (TEM) and scanning electron microscopy (SEM). ¹HNMR, DSC, XRD, etc. demonstrated that GA/CUR/HCPT-NPs had successful self-assembly, and had a great stability when stored at 4℃ within 7 days. The drug loading contents of GA, CUR, and HCPT in GA/CUR/HCPT-NPs determined by high-performance liquid chromatography (HPLC) were 57.19%, 39.17%, and 3.07%, respectively. Conclusion: In this study, GA/CUR/HCPT-NPs with appropriate size and uniform distribution through optimized anti-solvent precipitation method combined with dialysis method is successfully prepared, which lays a foundation for further experiments.