Among 25 isolates, Aspergillus fumigatus ASH (JX006238) was identified as a potent producer of homocysteine γ- lyase. The nutritional requirements to maximize the enzyme yield were optimized under submerged (SF) and solid-state fermentation (SSF) conditions, resulting in a 5.2- and 2.3-fold increase, respectively, after the last purification step. The enzyme exhibited a single homogenous band of 50 kDa on SDS-PAGE, along with an optimum pH of 7.8 and pH stability range of 6.5 to 7.8. It also showed a pI of 5.0, as detected by pH precipitation with no glycosyl residues. The highest enzyme activity was obtained at 37-40℃, with a Tm value of 70.1oC. The enzyme showed clear catalytic and thermal stability below 40℃, with T1/2 values of 18.1, 9.9, 5.9, 3.3, and 1.9 h at 30℃, 35℃, 40℃, 50℃, and 60℃, respectively. Additionally, the enzyme Kr values were 0.002, 0.054, 0.097, 0.184, and 0.341 S-1 at 30oC, 35oC, 40oC, 50℃, and 60℃, respectively. The enzyme displayed a strong affinity to homocysteine, followed by methionine and cysteine when compared with non-S amino acids, confirming its potency against homocysteinuriarelated diseases, and as an anti-cardiovascular agent and a specific biosensor for homocysteinuria. The enzyme showed its maximum affinity for homocysteine (Km 2.46 mM, Kcat 1.39 × 10-3 s-1), methionine (Km 4.1 mM, Kcat 0.97 × 10-3 s-1), and cysteine (Km 4.9 m M, Kcat 0.77 × 10-3 s-1). The enzyme was also strongly inhibited by hydroxylamine and DDT, confirming its pyridoxal 5`-phosphate (PLP) identity, yet not inhibited by EDTA. In vivo, using Swiss Albino mice, the enzyme showed no detectable negative effects on platelet aggregation, the RBC number, aspartate aminotransferase, alanine aminotransferase, or creatinine titer when compared with negative controls.