To investigate the potential functions of the hydrophobin-encoding gene Unigene0004454 from Ganoderma lucidum strain ZJ-1, gene cloning, bioinformatic analysis, promoter element prediction, and heterologous expression were performed. The results showed that the full-length genomic sequence of Unigene0004454 was 436 bp, with a cDNA length of 330 bp encoding a protein of 109 amino acids. Amino acid sequence analysis revealed the presence of the conserved cysteine pattern characteristic of hydrophobins and indicated a close evolutionary relationship with hydrophobins from Ganoderma leucocontextum and Ganoderma sinense. Promoter analysis demonstrated that the upstream regulatory region of Unigene0004454 contains core promoter elements (e.g., TATA box), multiple hormone-responsive elements (e.g., ABRE), and stress-responsive cis-elements (e.g., MBS), suggesting that this gene may be involved in strain growth, development, and stress responses. For heterologous expression, the recombinant plasmid pET-32a-Unigene0004454 was successfully constructed and transformed into E. coli BL21(DE3). SDS-PAGE and western blot analyses confirmed the expression of a recombinant protein with an apparent molecular weight of approximately 27 kDa, demonstrating the feasibility of heterologous expression of Unigene0004454. These findings provide a molecular basis for further investigation of the biological functions of Unigene0004454 and support the potential development of hydrophobin-based functional applications.