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[Objective] The present paper aimed to explore the effects of different nitrogen application rates on the gene levels of betaine and key enzyme contents in Lycium barbarum, and provide technical support for the quality generation cultivation of L. barbarum in Ningxia. [Method] In the experiment, Ningqi No. 1' was used as the experimental material, and the split-plot experiment design was used. The main area was two treatments of nitrogen fertilizer application depth A (A1: 0-20 cm, A2: 20-40 cm), and the sub-area was five treatments of nitrogen application amount B (CK: 0 kg/666.7 m², B1: 15 kg/666.7 m², B2: 30 kg/666.7 m², B3: 45 kg/666.7 m², B4: 60 kg/666.7 m²). Through the determination of related indicators during the growth period of L. barbarum and the high-throughput transcriptome sequencing analysis of L. barbarum samples with different nitrogen levels, the effects of nitrogen on betaine and key metabolic enzymes in L. barbarum were clarified. [Result] With the increase of nitrogen application rate, the contents of betaine and its synthesis-related enzymes in L. barbarum were basically on the rise. When the concentration reached 45 kg/666.7 m², the contents of the measured indexes began to decrease. A total of 98 264 genes were obtained by transcriptome sequencing, including 10 081 differentially expressed genes. GO annotation analysis showed that the annotated genes were mainly enriched in photosynthesis, apoplast, monooxygenase activity, chlorophyll, tetrapyrrole binding enzyme activity and other processes. After KEGG annotation, it was found that the differential gene signal patnway was significantly enriched in plant hormone signal transduction, plant pathogen interaction and ribosome. The differentially expressed genes related to betaine synthesis were analyzed, and it was found that the expression levels were up-regulated to varying degrees. Comprehensive analysis of the relationship between the content of the measured indicators and the results of gene expression levels showed that the up-regulation of betaine synthesis-related enzyme genes led to changes in betaine content in L. barbarum fruits. [Conclusion] The key enzymes of betaine synthesis are monooxygenase and betaine aldehyde dehydrogenase. With the increase of nitrogen application rate, the content of key enzymes in betaine synthesis and the number of related genes are up-regulated, resulting in the increase of betaine content in L. barbarum fruit. [ABSTRACT FROM AUTHOR]