A convenient and ultrasensitive self-powered aptasensing device based on an enzymatic biofuel cell (EBFC) was developed for myoglobin (Mb) detection. The self-powered aptasensor was composed of a functionalized biocathode with carbon nanotube-gold nanoparticle-aptamer (CNT-AuNP-aptamer) and a bioanode with carbon nanotube-gold nanoparticleglucose oxidase (CNT-AuNP-GOx). To ensure the performance of the self-powered aptasensor, the CNT-AuNP hybrid was selected as the electrode material because of its excellent biocompatibility, superior electrical conductivity, and chemical stability. Meanwhile, aptamer was assembled onto the CNT-AuNP functionalized biocathode through the Au−S bond. When the Mb was present, the aptamer could capture it on the biocathode via specific recognition, and the significant steric hindrance efficiently blocked the electronic transport for the redox probe [Fe(CN) 6 ] 3− at the biocathode, which caused a dramatic decrease of the open-circuit voltage (E OCV ). Promisingly, this self-powered aptasensor has particularly high sensitivity for Mb detection over a wide concentration spectrum of 0.1−1 × 10 4 ng mL −1 ; the detection limit is as low as 0.011 ng mL −1 (S/N = 3), and the sensor has excellent selectivity, reproducibility, and stability. In addition, the results of the relative standard deviation (RSD) (3.19−5.34%) and recovery (99.27−101.34%) indicated that the aptasensor can be applied to accurately assay Mb within complex biological matrices. As in situ Mb monitoring is of crucial clinical diagnostic significance, this work not only provides an intelligent method for ultrasensitive biosensing of Mb but also is a prospective prototype for a portable and in situ biomedical sensor.