A simple and ultrasensitive electrochemical biosensor employing a morpholino oligomer as capture probe and a cationic redox polymer as signal generator for direct detection of DNA is presented in this report. It is based on the immobilization of the morpholino oligomer on an indium-tin oxide (ITO) electrode and amperometric detection of target DNA by forming a DNA/cationic redox polymer bilayer on the ITO electrode. After hybridizing the morpholino capture probe (MCP) to the target DNA, the cationic redox polymer was introduced to the ITO electrodevia electrostatic interaction with the hybridized DNA. The deposited redox polymer exhibited excellent electrocatalytic activity towards the oxidation of ascorbic acid (AA), allowing for direct voltammetric and amperometric detection of DNA. Under optimized experimental conditions, a detection limit of 1.0 pM and linear current–concentration relationship up to 500 pM were obtained in amperometry. The resulting biosensors offered much better mismatch discrimination against mismatch sequences than their DNA counterparts.