Fusarium oxysporum is ubiquitous and can easily contaminate food during processing and storage, potentially producing T-2 toxin, which can pose a severe health risk to public health. Previous research on the presence of T-2 has focused on starch-rich foods, while protein- and amino acid-rich foods have received relatively little attention. In this study, the effects of amino acids on the growth of F. oxysporum and its T-2 production were investigated by gene deletion and complementation experiments. The results showed that amino acids, including aspartic acid, methionine, isoleucine, serine, phenylalanine, and cysteine, significantly inhibited the growth of F. oxysporum, while promoting T-2 synthesis, with cysteine having the most pronounced effect. The target of rapamycin complex 1 (TORC1) is a key pathway in response to a variety of amino acids, including cysteine. gtr2 and tap42 were found to be negative regulators of T-2 synthesis. The study highlights the elevated risk of T-2 production by F. oxysporum in cysteine-rich foods and the need to take appropriate measures to prevent and control the potential harm that such foods may present to public health. IMPORTANCE F. oxysporum and its T-2 contamination of food not only leads to food wastage but also poses a major food safety challenge to humans. The growth and T-2 production characteristics of F. oxysporum in high-protein substrates are considerably different from those in grains. Here, we show that the abundant free amino acids in a protein-rich food matrix are a key regulatory factor for the growth of, and toxin production by, F. oxysporum. Cysteine has the most pronounced effect on inhibiting mycelial growth and promoting T-2 synthesis through the TORC1 pathway. This implies that consumers tend to overlook T-2 contamination due to the poor growth of F. oxysporum in food rich in protein and amino acids, especially cysteine. Therefore, particular attention should be paid to the protection of those products.