Abstract: This study aimed to establish a genetic transformation system for Aureobasidium pullulans using an optimized Agrobacterium tumefaciens-mediated transformation (ATMT) technique to screen high-yield poly malic acid-producing strains. Key parameters, including the initial Agrobacterium concentration, pre-cultivation time, co-cultivation time, co-cultivation pH, acetosyringone (AS) concentration, and co-cultivation temperature, were optimized. The hygromycin phosphotransferase (hph) gene and enhanced green fluorescent protein (eGFP) gene were used as selection markers to identify high-yield poly malic acid-producing A. pullulans strains. The optimal transformation conditions were determined as follows: an initial Agrobacterium OD₆₀₀ value of 0.6, a pre-cultivation time of 8 hours, a co-cultivation time of 72 hours, a co-cultivation pH of 5.2, an AS concentration of 100 μmol/L, and a co-cultivation temperature of 22 ℃. Under these optimized conditions, a series of stable T-DNA insertion transformants were successfully constructed. Among them, the A. pullulans yield of high-yield mutant strains significantly increased, with the highest improvement reaching 74.59% compared to the wild-type strain. This study provides critical technical support for the strain optimization of A. pullulans industrial production and serves as a valuable reference for the genetic engineering of other industrial microorganisms.