Aquaculture waters often contain antibiotics, which inhibit the removal of ammonia nitrogen (NH₄⁺-N) by traditional microbial technologies. Thus, it is necessary to find potential microorganisms that can simultaneously tolerate to antibiotics and remove NH₄⁺-N. This study applied a novel isolated multi-drug-resistant yeast strain named Naganishia diffluens Mo to remove NH₄⁺-N in antibiotic-contaminated water. It was found the optimal conditions (glucose as carbon source, pH 7.0, 25 °C, 2 % inoculation, C/N ratio of 10) based on single-factor experiments for Naganishia diffluens Mo removing NH₄⁺-N could achieve 91.6 %. Response surface methodology further opitimized this condition as C/N ratio of 15, 24.9 °C, and pH 6.9, achieving 94.8 % of NH₄⁺-N removal, demonstrating great potentail to remove NH₄⁺-N. Whole-genome analysis indicated that Naganishia diffluens Mo likely removes NH₄⁺-N through ammonia assimilation, utilizing the GDH pathway and the GS-GOGAT metabolic pathway to incorporate NH₄⁺-N into biomass without converting it to nitrate (NO₃⁻) and nitrite (NO₂⁻). Besides, Naganishia diffluens Mo contains multiple antibiotic-resistance genes and genes reponsible for NO₃⁻ and NO₂⁻ removal, suggesting its great potential for N removal in antibiotic-contaminated wastewater.