A novel amperometric immunosensor for the determination of progesterone, based on direct attachment of antiprogesterone on a gold nanoparticles-modified graphite-Teflon composite electrode is reported. Competitive assay involving progesterone labeled with alkaline phosphatase (AP), and 1-naphtyl phosphate as the enzyme substrate, was employed. The product of the enzyme reaction, 1-naphtol was detected amperometrically at +0.3 V vs. Ag/AgCl. Variables involved in the immunosensor preparation such as the colloidal gold loading and the amount of anti-Prog immobilized, as well as in the immunosensing event (pH, Prog/Prog-AP ratio and volume) were optimized. A calibration plot was obtained for progesterone with a linear range between 0 and 30 ng mL−1, and a slope value, −1.2 nA ng−1 mL, which is remarkably higher than that reported for other progesterone biosensors. A good repeatability of the amperometric measurements, RSD=3.5% (n=10), and an acceptable fabrication reproducicility, RSD=8.2% (n=3), were obtained. The developed anti-Prog-Aucoll-graphite-Teflon immunosensor was applied to the progesterone determination in milk. Recovery at the 3.5 ng mL−1 progesterone concentration level was of 101±6% (n=7).