Trehalose, trehalose-6-phosphate synthase (TPS),and trehalose-6-phosphatase (TPP) have been reported to play important roles in plant abiotic stress and growth development. However, their functions in the flowering process of Rosa hybrida have not been characterized. In this study we found that, under a short photoperiod or weak light intensity, the content of trehalose in the shoot apical meristem of Rosa hybrida cv 'Carola' significantly decreased, leading to delayed flowering time. A total of nine RhTPSs and seven RhTPPs genes were identified in the genome. Cis-element analysis suggested that RhTPS and RhTPP genes were involved in plant hormones and environmental stress responses. Transcriptome data analysis reveals significant differences in the expression levels of RhTPSs and RhTPPs family genes in different tissues and indicates that RhTPPF and RhTPPJ are potential key genes involved in rose flower bud development under different light environments. The results of quantitative real-time reverse transcription (qRT-PCR) further indicate that under short photoperiod and weak light intensity all RhTPP members were significantly down-regulated. Additionally, RhTPS1a, RhTPS10, and RhTPS11 were up-regulated under a short photoperiod and showed a negative correlation with flowering time and trehalose content decrease. Under weak light intensity, RhTPS11 was up-regulated and negatively regulated flowering, while RhTPS5, RhTPS6, RhTPS7b, RhTPS9, and RhTPS10 were down-regulated and positively regulated flowering. This work lays the foundation for revealing the functions of RhTPS and RhTPP gene families in the regulation of rose trehalose.