Exposure to antibiotics during pregnancy can pose a systematic effect on human health. A few biomonitoring studies have demonstrated an extensive exposure of children to antibiotics, but there is still a lack of data for pregnant women. To assess the exposure of pregnant women to antibiotics and potential health risk, we investigated 536 pregnant women aged 16-42 years from two geographically different study sites in Eastern China in 2015. We measured 21 antibiotics of five categories (seven fluoroquinolones, three phenicols, four tetracyclines, three macrolides, and four sulfonamides) in urine using the isotope dilution ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The hazard index (HI) was calculated on the basis of estimated daily exposure dose and acceptable daily intakes. A total of 16 antibiotics were found in urine, with detection frequencies between 0.2 and 16.0%. Antibiotics were overall detected in 41.6% of urine, and two or more antibiotics were detected in 13.1% of urine. Ciprofloxacin, ofloxacin, and trimethoprim were most frequently detected in urine, with detection frequencies between 10 and 20%. The majority of the antibiotics tested had an estimated daily exposure dose less than 1 μg/kg/day, and 4.3% of pregnant women had a HI value of more than 1. These findings indicated that pregnant women were frequently exposed to antibiotics and some individuals were in the potential risk of adverse microbiological effects induced by antibiotics.