Rosa roxburghii Tratt. is a specific fruit with high nutritional value and antioxidative activities. However, the key metabolites and their biosynthesis are still unknown. Herein, a main cultivated variety, ‘Guinong 5’ (Rr5), was chosen to analyze the metabolomics of the three developmental stages of R. roxburghii fruit by liquid chromatography–tandem mass spectrometry (LC-MS/MS). A total of 533 metabolites were identified, of which 339 were significantly altered. Total phenols, flavonoids, and amino acids were significantly correlated to at least one in vitro antioxidant activity. The conjoint Kyoto Encyclopedia of Genes and Genomes (KEGG) co-enrichment analysis of metabolome and transcriptome was focused on amino acid, phenylpropanoid, and flavonoid biosynthesis pathways. The amino acid, phenolic acid, and flavonol biosynthesis networks were constructed with 32 structural genes, 48 RrMYBs, and 23 metabolites. Of these, six RrMYBs correlated to 9–15 metabolites in the network were selected to detect the gene expression in six different R. roxburghii genotypes fruits. Subsequently, 21 key metabolites were identified in the in vitro antioxidant activities in the fruits at various developmental stages or in fruits of different R. roxburghii genotypes. We found that four key RrMYBs were related to the significantly varied amino acids, phenolic acids, and flavonol derivatives in the network during fruit development and the key metabolites in the in vitro antioxidative activities in the fruits of six R. roxburghii genotypes. This finding provided novel insights into the flavonoid, polyphenol, and amino acid synthesis in R. roxburghii.