Background: There is a substantial decline in first-service-pregnancy-rate in dairy cows. In this regard, future prospects are to measure milk hormones on-farm and progesterone levels in milk are not enough to precise ovulation unless connected to other data. The objectives of this study were to investigate whether 17β-estradiol could be measured from individual cow milk samples using a commercially available non-radiolabelled enzyme immunoassay kit (EIA) with no previously reported milk application, and whether those detections could precisely illustrate 17β-estradiol pre-ovulation peak in spite of its limited concentration and short manifestation in milk. Results: Milk sample treatments for progesterone and 17β-estradiol EIA measurements are described. Hormonal profiles from daily milk samples of six different cows were reported and 17β-estradiol pre-ovulation peak was visualized in all cases. Heat detection was possible by EIA using one every 2 days milking samples in almost all studied cases. Only in one case, morning and afternoon milking samples were required to visualize the 17β-estradiol pre-ovulation peak. Conclusions: 17β-estradiol EIA quantification in raw milk is a reliable, rapid, economic and a precise method to describe cow heat along with EIA progesterone determination.