Trehalose6phosphate synthase gene TPS is one of the key genes for the synthesis of trehalose. In this study, the homologue of TPS5 was isolated from Petunia hybrida, which was named PhTPS5. The open reading frame（ORF）of PhTPS5 was 2595 bp and encoded a protein of 864 amino acids. The molecular formula of PhTPS5 protein was assumed to be C₄₃₆₃H₆₈₂₅N₁₁₇₃O₁₂₈₉S₃₇. Tissuespecific expression analysis showed that PhTPS5 had the highest expression level in roots and the lowest in leaves. After 6 h of decapitation, PhTPS5 expression was significantly promoted, and the expression level decreased significantly at 24 h. The application of auxin to the top could effectively inhibit the regulation to PhTPS5. The expression level of PhTPS5 was significantly upregulated after 6 h of cytokinin treatment, and decreased with the increase of treatment time. This study laid a theoretical foundation for further revealing the regulatory mechanism of the TPS pathway in the shoot branching of petunia.