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We propose a novel technique to pattern active proteins on a glass substrate using a perforated poly-dimethylsiloxane sheet (PDMS sieve). It is fabricated by spin-coating PDMS on a pyramidal shaped mold. Proteins such as FITC-albumin and F1 -ATPase biomolecular motors are successfully spotted within 5×5 μm2 in an array. The patterned spots are perfectly isolated, which ensures the elimination of non-specific binding of the protein to the undesired area. Furthermore, it is confirmed that the proteins maintain their activity after the patterning by observing the rotation of the ATPase. The possibility of the patterning of different proteins is indicated by selectively spotting three kinds of fluorescent micro-beads.