Polystyrene microspheres with diameter of 350 nm were prepared, and then used to arrange on the surface of glassy carbon electrode (GCE) as structure-directing template. By successive cyclic sweeps between -1.0 V and 2.0 V in pH 7 phosphate buffer containing 10 mM cysteine, polycysteine film was electrodeposited on polystyrene microspheres-arranged GCE. After removing polystyrene template, a three-dimensionally ordered macroporous (3-DOM) polycysteine film was achieved, as confirmed by scanning electron microscopy measurements. Electrochemical responses of K-3[Fe(CN)(6)] probe indicated that 3-DOM polycysteine film-modified GCE exhibited larger active area, compared with GCE, polycysteine film-modified GCE and electrochemically oxidized GCE. The application of 3-DOM polycysteine film in electrochemical detection of 4-chlorophenol was studied. Due to ordered porous structures, the 3-DOM polycysteine film-modified GCE displayed signal enhancement effects, and enhanced the oxidation peak currents of 4-chlorophenol. As a result, a sensitive electrochemical method was developed for the detection of 4-chlorophenol, and the detection limit was 1.67 x 10(-8) M. This new method was used to detect 4-chlorophenol in water samples, and the value of recovery was over the range from 99.6% to 107%. (C) 2014 Elsevier Ltd. All rights reserved.