Ultrafiltration experiments for the chiral separation of racemic tryptophan, phenylglycine and phenylalanine were investigated through immobilized DNA membranes having various pore sizes. l-tryptophan preferentially permeated through immobilized DNA membranes with a pore size<2.0 nm (molecular weight cut-off (MWCO)<5000) while d-tryptophan preferentially permeated through immobilized DNA membranes with a pore size>2.0 nm (MWCO>5000). These results are completely opposite tendency in the ultrafiltration of racemic phenylalanine through the immobilized DNA membranes. This may be originated from the different interaction between DNA and tryptophan compared to that between DNA and phenylalanine. However, in both cases 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. Chiral separation models were proposed from using the chiral separation results of racemic amino acids, preferential adsorption of amino acid enantiomers and EPMA results.