A novel, ultra-high sensitivity electrochemical aptamer biosensor (EAB) was fabricated by immobilising gold nanoparticles (Au) on a nano-confined interface of N-doped carbon nanofibers/carbon fibers (N-CNFs/CFs). Gold nanoparticle-thiol (Au–S) conjugates, coupled with aptamer-specific recognition technology, were used to immobilise aflatoxin B1 (AFB1). The nanoconfined interface of N-CNFs/CFs provides more binding sites for Au with its unique spatial structure and electroactive surface area, enhancing the electrochemical performance of the matrix. Compared to the existing sensor detection limit, the limit of detection(LOD) of the EAB was approximately 6.4 pg/mL. The dynamic detection ranged from 10.0 to 1.0 × 108 pg/ mL. Furthermore, AFB1 was also successfully detected in Chinese Materia Medica decoction pieces(CMMDP) using the prepared EAB, with recoveries ranging from 96.18 to 112.87%. These results demonstrate the proposed EAB’s potential as a reliable tool for rapid and efficient detection of AFB1 in complex matrices.