As an important subclass of metal-organic frameworks (MOFs), homochiral MOFs show special ability of asymmetric catalysis and enantioselective separation via the open chiral channels or cavities. A new homochiral MOF membrane [Ni 2 (mal) 2 (bpy)]•2H 2 O (Ni-MB) was synthesized by a secondary growth technique, in which high energy ball milling was employed to prepare nanosized MOF seeds, and their synthesis condition were optimized. X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), and thermal gravimetric analysis were used to measure physicochemical properties of the MOF crystals and membranes. In particular, the homochiral MOF membranes were characterized by nano-indentation/scratch technique with high-resolution mechanical property analysis. The SEM results indicated that the membrane was continuous and integrated without observable defects; the permeation measurements of single gas show that the permeate flux of nitrogen was independent on the transmembrane pressure, further confirming the absence of macroporous defects in the as-prepared chiral MOF membranes. It is expected that the proposed chiral MOF membrane can be potentially applied to enantioselective separation in the future.