A new green competitive ELISA for aflatoxin M1 quantification in raw milk was developed. This diagnostic tool is based on an anti AFM1 mAb produced by plant molecular farming in alternative to classical systems. Our assay, showing an IC50 below 25 ng/L, fits with the requirements of EU legislation limits for AFM1 (50 ng/L). Optimal accuracy was achieved in correspondence of the decision levels (25 and 50 ng/L), and the assay enabled AFM1 quantification in the range 5–110 ng/L, with limit of detection 3 ng/L. Moreover, to evaluate a real applicability in diagnostics, raw milk-spiked samples were analysed, achieving satisfactory recovery rates of AFM1. In conclusion, an efficient and ready-to-use diagnostic assay for the quantification of aflatoxin M1 in milk, based on a plant-produced recombinant mAb, has been successfully developed.