CYTOSINE Deaminase (CDA) is a non-human enzyme converting cytosine to uracil, with significant implementation on various cancer therapeutic approaches especially prodrug mediated therapy using 5-fluorocytosine into 5-fluorouracil. However, the lower catalytic/ thermal stability and higher antigenicity of this enzyme are the main challenges for further its clinical applications, thus, screening for thermostable enzyme with higher turnover activity was the objective of this study. Among the recovered thermotolerant fungal isolates, Aspergillus fumigatus has been selected as potent CDA producer. Upon nitrogen starvation, the yield of intracellular CDA by A. fumigatus was significantly increased by about 5 folds, comparing to control. The enzyme was purified from thermotolerant A. fumigates into its electrophoretic homogeneity by ion-exchange and gel-filtration chromatography by 4.4 purification folds and 29.9% yield, respectively, with molecular subunit structure 48 kDa under denaturing-PAGE. The purified enzyme showed an optimum pH 7.0, optimum reaction temperature 37°C. The maximum affinity (Km) and reaction velocity (Vmax) of purified CDA was 0.08 mM and 400 μmol/min/mg on cytosine as substrate. At 37°C, the half-life time (T1/2) of purified CDA was about 8 h, ensuring the structural/ catalytic thermal stability of this enzyme. Based on these preliminary results, A. fumigatus CDA could be a scaffold for further in vivo studies on cancer prodrug-mediated therapies, or gene therapy applications.