A light source plays a pivotal role in a photofuel cell (PFC)-based self-powered biosensor. Although a visible light source has been extensively employed to drive a PFC, it still has some drawbacks for biosensing due to its relatively high energy. Herein we constructed a PFC-based aptasensor using near-infrared (NIR) light as the irradiation source. To achieve an efficient absorption of the NIR light, NaYF4:Yb,Er upconversion nanoparticles (UCNPs) that could convert low-energy incident light into high-energy radiation were combined with Bi2S3 nanorods (UCNPs/Bi2S3) to serve as the photoactive materials. The PFC was comprised of a UCNPs/Bi2S3 photoanode and a Pt cathode, which could generate electrical output under NIR light irradiation to provide the self-powered sensing signal without the supply from an external power source. The aflatoxin B1 (AFB1) binding aptamer was immobilized on the photoanode to serve as the recognition element. The detection of AFB1 was based on the competition between the interaction of aptamer with AFB1 analyte and the hybridization of aptamer with Au nanoparticles-labeled DNA sequence (AuNPs-cDNA). Under optimum conditions, the proposed aptasensor presented good sensitivity and high specificity for AFB1 detection in the concentration range from 0.01 to 100 ng·mL-1, with a detection limit of 7.9 pg·mL-1. Moreover, the developed sensor was applied to an assay of AFB1 in flour samples with a desirable accuracy and precision.