A new model ELISA, based on two monoclonal antibodies, was developed for the quantification of fatty acid synthase (FAS). In this sandwich assay, a monoclonal antibody M6 was used as a capture on Nunc MaxiSorp ELISA/EIA Modules and another monoclonal antibody M3, labeled with biotin, was used as a detection antibody. More than 10 molecules of biotin were labeled on the anti-FAS monoclonal antibody using modified biotinylation conditions. The within- and between-run CVs were less than 10%, and the detection limit was 3.22 ng/mL. Recoveries were 98.54-121.95%, averaging 106.05%. The average FAS concentration obtained from the total 55 healthy volunteers blood was 4.07 +/- 1.81 ng/mL, 4.25 +/- 2.14 ng/mL in women (n = 37) and 3.70 +/- 0.74 ng/mL in men (n = 18). When compared with the previously developed polyclonal-monoclonal ELISA, a different pattern of FAS levels was observed in the supernatant of two cultured breast cancer cell lines in a time course study and there was no linear correlation between the two assays using 215 human blood samples. Thus, this new model FAS-ELISA could be used as an independent assay in measuring clinical samples. In summary, this monoclonal-monoclonal FAS-ELISA is sensitive, accurate, and precise in quantification of fatty acid synthase and has potential as a complementary tool in testing clinical samples.