Abstract: In this study, we collected Lycium barbarum L. from different areas in Ningxia and determined the contents of 17 protein amino acids. Based on the amino acid pattern recommended by FAO/WHO, we comprehensively evaluated the nutritional value of protein amino acids and regional differences using indicators, such as amino acid score (AAS), chemical score (CS), amino acid ratio coefficient (RC), and amino acid score of ratio coefficient (SRC), combined with correlation, principal component, and cluster analyses. Our results revealed that Ningxia L. barbarum L. contained seven, nine, and five kinds of essential, medicinal, and flavor amino acids (EAA, MAA, and FAA), respectively. The total amino acid (TAA) and EAA contents ranged from 7.500 to 9.490 and 1.545 to 2.276 g/100 g, respectively. The EAA/TAA and Essential-to-Non-Essential Amino Acid (EAA/NEAA) ratios were between 19.38%~26.11% and 24.03%~31.55%, respectively. Both the EAA/TAA and EAA/NEAA values were lower than the ideal protein reference values (i.e., 40 % and 60 %, respectively) given by the FAO. The total medicinal amino acid (TMAA) content ranged from 4.314 to 5.886 g/100 g, accounting for 56.59%~62.40% of the TAA, indicating high medicinal value. The total FAA content ranged from 1.557 to 2.341 g/100 g, accounting for 19.53%~26.04% of the TAA. We observed no significant differences (P>0.05) in the TAA, EAA, NEAA, MAA, and FAA contents among the different Ningxia producing areas, except for the significantly different TAA content between Tongxin and both Zhongwei and Guyuan. However, we detected significant differences (P<0.05) in the EAA/TAA and EAA/NEAA ratios as well as in the total TMAA and FAA contents. The key amino acids we identified were Gly, Asp, Glu, Met, Thr, Val, Phe, Leu, Ile, and Lys, and the combination of Cysteine and Methionine (Cys+Met) was the first limitation. The AAS, CS, and SRC values ranged between 58.2~102.8, 66.5~98.6, and 71.1~84.9, respectively. L. barbarum L. from Zhongwei, Guyuan, Yinchuan, Haiyuan, and Zhongning regions exhibited superior comprehensive amino acid quality, with SRC evaluation results indicating a more balanced composition closer to the FAO/WHO-recommended pattern. Cluster analysis categorized the L. barbarum L. from Ningxia producing areas into four distinct groups: 1) Qingtongxia and Tongxin; 2) Yueyahu, Helan, and Hongsibao; 3) Haiyuan and Zhongning; 4) Guyuan, Yinchuan, and Zhongwei. The experimental results effectively reflect the regional differences in protein amino acids and the nutritional value of Ningxia Lycium barbarum L., providing a valuable reference for deep processing.