Aspergillus flavipes L-methionine γ-lyase (MGL) has been authenticated as a powerful anticancer agent towards various solid tumors, however, the catalytic efficiency and stability of this enzyme remains the main challenge for its further in vivo applications. Thus, the objective of this study was to enhance the catalytic efficiency, structural stability of A. flavipes MGL, in addition to boost their anticancer activity, via conjugation with β-cyclodextrin. The purified A. flavipes MGL was (38.1 μmol/mg/min) was conjugated with β-cyclodextrin, with immobilization yield 80%. The conjugation process of MGL with β-cyclodextrin was verified from the FTIR analysis, molecular docking analysis, ensuring the covalent conjugation process via the hydrogen, and hydrophobic interactions with the cyclodextrin hydroxyl groups and MGL surface amino acids residues. The free and CD-MGL have the same optimum reaction temperature 37 °C, reaction pH 7.5 and pH stability pH 6.5–8.0. The CD-MGL conjugates had a significant stability to proteinase K and trypsin digestion. The affinity of CD-MGL was increased by ~ 2 folds to L-methionine (KM 3.1 mM), compared to the free one (KM 7.2 mM), as well as the catalytic efficiency of MGL was increased by 1.8 folds upon cyclodextrin conjugation. The higher affinity of CD-MGL for L-methionine might be due to re-orientation of the MGL to bind with the substrate by multiple interactions hydrogen, hydrophobic and covalent bonds compared to the free one. The thermal stability of MGL was increased by ~ 2 folds for the tested treatments, upon cyclodextrin conjugation. The in vitro anticancer activity of CD-MGL was enhanced by 2 folds against the HCT-116 (IC50 value 13.9 μmol/mg/min) and MCF7 (IC50 value 9.6 μmol/mg/min), compared to the free MGL (~ 21.4 μmol/mg/min). The enzymes displayed a significant activity against the proliferation of Ehrlich ascites carcinoma in vivo, with