Background Ergometrine maleate injection as a first-line drug to prevent postpartum hemorrhage. To investigate the safety of ergometrine maleate injection and breastfeeding. Methods Pregnant women were recruited into this study according to the inclusion and exclusion criteria and their peripheral blood and breast milk were collected for the first time (some pregnant women have no milk at this time). After the delivery of the fetus, some parturients were further excluded according to the exclusion criteria. The recruited women were randomly divided into the control group and the treat group. The peripheral blood was collected in the control group at 2h and 12h after delivery, respectively, and the peripheral blood and breast milk were collected in the treat group at 2h, 4h, 6h and 12h after delivery, respectively. The concentrations of ergometrine maleate in maternal plasma and breast milk at different periods were detected by ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS), and prolactin levels at different time gradients after delivery were detected with chemiluminescence. Results Sixty-four women were finally recruited, with 32 in the treat group and 32 in the control group. The final concentration of ergometrine maleate injection reached the peak at 2h after administration and basically decreased to the pre-administration level at 12h after administration in both serum and breast milk. The RID was 4.5762% (2.5146%, 7.8638%) and 0.1842% (0.0000%, 1.1838%) at 6h and 12h after administration, respectively. Ergometrine maleate injection does not affect the PRL concentrations in peripheral blood at 2h and 12h. Conclusion The results suggest that breastfeeding at 12h after ergometrine maleate injection is relatively safe. In this study, ergometrine maleate injection was not found to inhibit the concentration of PRL in the peripheral blood, and no serious adverse reactions were found.