Wolfberry, regarded as a nutritious fruit, has garnered significant attention in the food industry due to potential health benefits. However, the tissue-specific distribution and dynamic accumulation patterns of nutritional metabolites such as flavonoids are still unclear. In this study, a novel spatial metabolomics framework was developed, incorporating instrumental optimization, metabolite identification, molecular network analysis, metabolic pathway mapping, and machine learning-based imaging. Using DESI-MSI, this approach enabled rapid, non-destructive, in situ analysis of wolfberry metabolites with enhanced sensitivity and spatial resolution. Detailed insights into chemical and spatial changes during ripening were obtained, with a focus on flavonoids. The visualization of the flavonoid biosynthetic pathway highlighted the impact of C-3 hydroxylation on flavonoid redistribution. Furthermore, a classification model achieved a prediction accuracy exceeding 99 %, consistent with metabolic network analyses. This framework provides a powerful tool for plant metabolomics, facilitating the exploration of functional components and metabolic pathways.