Abuse of chloramphenicol (CAP) could cause serious side effects to human health. Therefore, it is necessary to detect CAP residues in animal sourced food effectively. Here, the superiority (better stability, higher Apt-CAP loading efficiency, and higher CAP binding associated conformational change, etc.) of tetrahedral structure to double-chained structure for developing aptasensor was evaluated. Then, a self-assembled DNA tetrahedral structure-based electrochemical aptasensor targeting CAP was developed. Under the optimized conditions, the aptasensor exhibited high sensitivity toward CAP with a limit of detection (LOD) of 0.0676 ng/mL (linear range 0.19-387.76 ng/mL), and high selectivity against the structural analogs of CAP. Moreover, the recovery rate of CAP from spiked milk samples ranged from 100.57% to 101.69%, and only USD 1.34 is needed for detecting CAP in 1 sample. These results suggested the application potential of this aptasesor for detecting CAP in animal sourced foods.