A sensitive electrochemical biosensor based on Aflatoxin-Oxidase (AFO) was developed for detection of sterigmatocystin (ST). The enzyme was immobilized on chitosan-single-walled carbon nanotubes (CS-SWCNTs) hybrid film, which attached to the poly-o-phenylenediamine (POPD)-modified Au electrode. The fabricated procedures of the biosensor were characterized with atomic force microscopy (AFM), fourier transform-infrared spectroscopy (FT-IR), and electrochemical impedance spectroscopy (EIS). The cyclic voltammetric results of the biosensor indicated that AFO exhibited a surface-controlled and quasi-reversible electrochemical redox behavior with a formal potential of -0.436 V (vs. Ag/AgCl) in 0.1 mol/L PBS (pH 7.0), which resulted from the direct electron transfer between entrapped AFO and the underlying electrode. The enzymatic electrode exhibited an excellent electrocatalytic response to ST. The linear range of ST determination was from 10 ng/mL to 310 ng/mL with correlation coefficient of 0.997, the detection limit was 3 ng/mL (S/N=3), and the response time was less than 10 seconds. The apparent Michaelis-Menten constant () was estimated to be 7.13 mmol/L. The biosensor had the advantages of good repeatability and stability, remaining 85.6% of its original current value after storage at 4oC for a month, and the RSD for 11 replicate determination of 20 ng/mL ST was 3.9%. This AFO/CS-SWCNTs/POPD/Au modified electrode showed high selectivity and sensitivity in real sample analysis, giving values of recovery in the range of 87.6%-105.5%. The proposed method can be applied to the determination of ST in real samples with satisfactory results.