Abstract: The study aimed at discussing trehalose synthesis-related enzyme genes of Flammulina velutipes and their functions,which could lay the foundation for revealing the changing rule during growth and development process. The sequence of tps1 gene was analyzed and relative quantitative expression changes of tps1 and tps2 in mycelia of Flammulina velutipes strain Dan 3 under temperature variations were measured by using real-time PCR. Results showed that open reading frame of tps1 gene contained 1455 bp encoding a polypeptide of 484 amino acid residues. After analysis of the section of 2000 bp upstream of initiator codon,it was found that the promoter contained typical regulatory elements,STRE(C4T) and HSE(AG4). The real-time PCR analysis showed that the relative expression of tps1 gene and tps2 gene both significantly(p<0.05) increased after 2 hours management of relatively high temperature 37 ℃ and relatively low temperature 4 ℃. The results confirmed that the expression of the tps1 and tps2 gene was regulated by temperature at transcriptional level.