Rosa rugosa is an important ornamental and edible species that is valued for its floral colors and essential oils in the cosmetic and pharmaceutical industries. Carotenoids, beyond their health-promoting roles, function as accessory pigments that influence petal coloration, flower quality, and stress responses. However, their accumulation patterns and molecular biosynthesis in R. rugosa remain poorly understood. Here, UPLC-APCI-MS/MS analysis across three developmental stages (bud, semi-open, and full bloom) revealed stage-specific carotenoid accumulation, with phytoene and phytofluene markedly increasing at the semi-open stage. In total, 11 carotenoids were identified, comprising four carotenes and seven xanthophylls. Differential accumulation of metabolites (DAMs) analysis indicated shifts in compounds, including (E/Z)-phytoene, phytofluene, and β-carotene across stages. Genetic complementation assays in Escherichia coli and transient overexpression in rose petals confirmed that RrPSY1 functions as a phytoene synthase. qRT-PCR results showed its upregulation under salt treatment, suggesting a role in enhancing stress tolerance through carotenoid-mediated antioxidant protection. Furthermore, sub-cellular localization experiments confirmed plastid targeting of RrPSY1. Together, these findings clarify the role of RrPSY1 in carotenoid biosynthesis and provide a foundation for future studies on metabolic regulation and biosynthesis of carotenoids in R. rugosa.