Photofuel cells (PFCs) that integrate “clean-solar-to-electric energy” and “sensing platform” can enable next-generation biosensors for diverse health care and environmental applications. However, the inferior performance of photoelectrodes often results in insufficient solar-to-power conversion for reliable molecular detection. To overcome this problem, we developed a PFC-based genosensor by combining a Ti3C2Tx|CdS photoanode and a CuInS2 photocathode to generate significant output power for the reliable detection of miRNA-21. The innovative photoanode design involves (i) the insertion of CdS nanorods into multi-layered Ti3C2Tx to expand the interlamellar space from ~0.98 to ~1.34 nm for accommodating more biomolecules to improve the sensor sensitivity; (ii) the formation of the nanoscale Schottky junction to enhance photo-generated charge separation and photoelectric conversion rates. The hybridization of capture DNA, assistant DNA and miRNA-21 on the photoanode led to the initial quenching of output signal of prepared PFC. As a mimic enzyme, manganese porphyrin was intercalated into the conjugated DNA double chains to catalyze a precipitation reaction on the photoanode surface, which resulted in a further signal quenching. Due to the combination of specially designed PFC with dual signal quenching, the proposed PFC-based genosensor exhibited a dynamic detection range of 1 fM to 0.01 μM with a LOD of 0.053 fM for detection of miRNA-21.