A flow-injection immunoassay (FI-IA) method with amperometric detection for aflatoxin M1 (AFM1) determination in milk has been developed. The first step consists in an incubation of the sample containing AFM1 (Ag) with fixed amounts of anti-AFM1 antibody (Ab) and of the tracer (Ag∗, AFM1 covalently coupled to HRP) until equilibrium is reached. In this mixture a competition occurs between Ag and Ag∗ for the Ab. The mixture is then injected into a flow system where the separation of the free tracer (Ag∗) and the antibody-bound tracer (AbAg∗) is performed in a column with immobilized Protein G. The antigen–antibody complexes are retained in the column due to the high affinity of the Protein G for the antibody. The activity of the eluted enzyme label is then amperometrically detected.The immunoassay was optimised relative to conditions for antibody–antigen incubation (pH, incubation time, ionic strength, temperature) and enzymatic label detection. This method showed a dynamic concentration range between 20 and 500 ppt AFM1, a low detection limit (11 ppt), good reproducibility (RSD < 8%) and a high throughput (six samples per hour in triplicate). Different milk samples were analysed and the results were in good agreement with those obtained by HPLC using the AOAC 2000.08 method.