Herein a photoelectrochemical (PEC) and electrochemical (EC) dual-mode biosensor with cationic N,N-bis(2-(trimethylammonium iodide)propylene)perylene-3,4,9,10-tetracarboxydiimide (PDA+)-decorated multifunctional DNA spheres in situ generated on an electrode was proposed for sensitive and accurate detection of miRNA-141. By employing a target-related ternary "Y" structure cleavage cycling reaction, the target DNA was converted into massive output DNA anchored on a TiO2 substrate, and hence triggering the rolling circle amplification (RCA) reaction. Upon addition of magnesium ions and PDA+, the long DNA tails of the RCA product were condensed in situ to form multifunctional DNA spheres. Notably, the distance between DNA spheres and TiO2 substrate was short, thus forming an effective PDA+-TiO2 sensitization structure with fast electron transfer for acquiring an extremely enhanced PEC signal with assistance of ascorbic acid (AA). Meanwhile, cationic PDA+ with a large planar π-π skeleton enabled favorable redox-activity and substantial loading on DNA spheres, directly producing an obviously well-defined cathodic peak for implementing EC biodetection on the same sensing platform. This approach not only avoided difficult assembly of diverse signal indicators but also significantly improved the sensitivity by utilizing cleavage cycling amplification and RCA strategies. Moreover, the distinct dual-response signals from two different transduction mechanisms and independent signal transduction can mutually support accuracy improvement. As a result, detection ranges of 0.1 fM to 1 nM for PEC and 2 fM to 500 pM for EC were obtained for miRNA-141, providing a universal and efficient biosensing method with promising applications in bioanalysis and early disease diagnosis.