The Lycium ruthenicum Murr. fruit is a widely used nutritional food that contains various bioactive components such as anthocyanin and spermidine derivatives. In the present study, ultra-high-performance liquid chromatography with tandem mass spectrometry was utilized to profile the metabolic dynamics of four developmental stages of Lr fruit. A total of 49 compounds, including anthocyanin, alkaloids, hydroxycinnamic acid derivatives, flavonoids, and amino acids, were tentatively identified. Principal component analysis distinguished the fruit at four developmental stages using 15 potential marker compounds. Pearson correlation analysis suggested that anthocyanin and spermidine derivative hexoses had a strong positive correlation coefficient. A glucosyltransferase (HG27071) was confirmed to glucosylate both anthocyanidin and spermidine derivative in vitro. Our results provide insight into the metabolic linkages among bioactive components in Lr fruits. The glucosyltransferase identified in this study will promote its potential use in functional foods and natural pigment resources.