γ-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the central nervous system, exerts physiological functions such as sedation, sleep promotion, and anxiolysis by attenuating neuronal excitability. In this study, a high-GABA-producing Saccharomyces cerevisiae strain was engineered for lychee wine fermentation. By utilizing CRISPR-Cas9 genome editing technology to knock out UGA2, a critical gene encoding succinate semialdehyde dehydrogenase involved in GABA catabolism, the recombinant strain S. cerevisiae SY-A2 was successfully constructed. When applied to lychee wine production, the SY-A2 strain yielded a GABA concentration of 943.85 mg·L-1, representing significant increases of 26.29% and 17.32% compared with the parental strain SY-0 (747.34 mg·L-1) and the UGA1-deleted strain SY-A1 (804.53 mg·L-1), respectively. To further maximize GABA accumulation, the fermentation process using strain SY-A2 was optimized via single-factor tests and response surface methodology based on a Box-Behnken design. The optimal fermentation parameters were determined as follows: a temperature of 30.50 °C, an inoculation size of 2.29×107 cells·mL-1 and a glutamate supplementation of 53 mg·L-1. Under these optimized conditions, the GABA concentration in the lychee wine reached 1459.87 mg·L-1. These findings establish an efficient bioengineering strategy and a robust technical framework for producing GABA-enriched functional fruit wines.