Highly stable circulating microRNAs (miRNAs) are currently recognized as a novel potential biomarker for clinical cancer diagnosis in the early stage. However, limited by its low concentration, high sequence similarity, as well as the numerous interferences in body fluids, detection of miRNA in whole blood with sufficient selectivity and sensitivity is still challenging. Herein, we reported the integration of entropy-driven toehold-mediated DNA strand displacement (ETSD) reaction with magnetic beads (MB) toward the energy-transfer-based photoelectrochemical (PEC) detection of the prostate carcinoma (PCa) biomarker miRNA-141 in a real blood sample. In this protocol, the ETSD reaction was divided into two steps, and cooperated with magnetic separation, target extraction and amplification could be realized in a single test and ultrasensitive detection of miRNA-141 could be achieved in undiluted whole blood sample. This work proposed a new solution for sensitive biomolecular detection in a complex biological milieu and exhibited great promise for future clinical cancer diagnosis.