The advantages of simple structure and easy portability inherent to self-powered biosensors have broad application prospects in clinical diagnosis and implantable medical devices. In light of the limitations of the clinical detection sensitivity, this study devised and fabricated a three-dimensional (3D) cubic structure of MoSS that was self-assembled by 2D nanosheets. The multi-dimensional structure design provides a broad binding site for the incubation of biological probes and the loading of biological enzymes, which can accelerate the electron transfer rate and effectively improve the detection sensitivity. On this basis, a self-powered biosensor platform was constructed for the detection of miRNA-499, employing the catalytic hairpin self-assembly (CHA) signal amplification strategy. This strategy has the potential to enhance the accuracy of detection and circumvent the occurrence of false positive results. The experimental results demonstrate that the self-powered biosensor exhibits a detection limit (LOD) of 0.12 fmol/L (S/N = 3) within a wide linear range of 1 fmol/L to 10 nmol/L. Furthermore, the sensor platform exhibits excellent specificity, stability, and reproducibility. Moreover, the test signal is transmitted via Bluetooth to the smartphone interface, thus meeting the requirements for portable, realtime detection.