In the current studies, we investigated the use of immobilized DNA membranes for chiral separation. D-Phenylalanine preferentially permeated through immobilized DNA membranes with a pore size <2.0nm (molecular weight cut-off (MWCO)<5000) while L-phenylalanine preferentially permeated through immobilized DNA membranes with a pore size >2.0nm (MWCO>5000). The pore size of the immobilized DNA membranes regulated preferential permeation of the enantiomer through the membranes. The immobilized DNA membranes are categorized as channel-type membranes and not as affinity membranes. We further propose a multi-stage cascade for chiral separation by ultrafiltration. When the two types of the membranes were used in a cascade filtration system, there was no change of concentration ratio between L-enantiomer and D-enantiomer in the feed solution at each stage. Only two membranes were necessary in a multi-stage processes if both membrane types were used.