Aflatoxin M1 (AFM1) is a toxic metabolite of the fungal product aflatoxin found in milk. For food safety concern, maximum residual limits of AFM1 in milk and dairy products have been differently enforced in many countries. A suitable detection method is required to screen a large number of product samples for the AFM1 contamination. In this study, monoclonal antibodies (MAbs) against AFM1 were generated using a conventional somatic cell fusion technique. After screening, five MAbs (AFM1-1, AFM1-3, AFM1-9, AFM1-11, and AFM1-17) were obtained that showed cross-reactivity with aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) but with no other tested compounds. An indirect competitive enzyme-linked immunosorbent assay (ELISA) using a partially purified MAb and antigen-coated plates yielded the best sensitivity with the 50% inhibition concentration (IC50) and the limit of detection (LOD) values of 0.13 ng/mL and 0.04 ng/mL, respectively. This indirect competitive ELISA was used to quantify the amount of fortified AFM1 in raw milk. The precision and accuracy in terms of % coefficient of variation (CV) and % recovery of the detection was investigated for both intra- (n = 6) and inter- (n = 12) variation assays. The % CV was found in the range of 3.50-15.8% and 1.32-7.98%, respectively, while the % recovery was in the range of 92-104% and 100-103%, respectively. In addition, the indirect ELISA was also used to detect AFM1 fortified in processed milk samples. The % CV and % recovery values were in the ranges of 0.1-33.0% and 91-109%, respectively. Comparison analysis between the indirect ELISA and high performance liquid chromatography was also performed and showed a good correlation with the R2 of 0.992 for the concentration of 0.2-5.0 ng/mL. These results indicated that the developed MAb and ELISA could be used for detection of AFM1 in milk samples.