<p>The development of a high-efficiency expression system is crucial for advancing the molecular breeding and synthetic biology of <i>Tremella fuciformis</i>. Here, the system is reported through the identification of potent endogenous promoters and the systematic optimization of an <i>Agrobacterium tumefaciens</i>-mediated transformation protocol. Key parameters for transformation were established, including optimal antibiotic concentrations (12.5&#xa0;µg/mL hygromycin, 250&#xa0;µg/mL cefotaxime) and the critical yeast-like cells (YLCs) density (OD₆₀₀ 0.6–0.8) for co-culture. In addition, scanning electron microscope (SEM) observation revealed that surface damage on YLCs treated with brown aluminum oxide facilitated T-DNA transfer from <i>A. tumefaciens</i> and resulted in a 2.25-fold increase in the number of transformants. Central to this system, <i>gene_sp10042040.1</i> (<i>P4</i>), <i>gene_sp10011850.1</i> (<i>P5</i>) and <i>gene_sp10036670.1</i> (<i>P7</i>) were validated in transformed YLCs, where they drove the expression of the e<i>GFP</i> to levels 67.8-fold, 11.8-fold, and 7-fold higher than the control wild-type strain, respectively. Strikingly, these novel promoters outperformed the constitutive <i>gpd</i> promoter, with an increase in expression efficiency of up to 1476.74%. This integrated expression system, comprising an optimized transformation workflow and a set of superior endogenous promoters, provides a powerful platform for genetic manipulation and metabolic engineering in <i>T. fuciformis</i>.</p> Graphical Abstract <p></p>