The development of reference values of trace elements is recognized as a fundamental prerequisite for the assessment of trace element nutritional status and health risks. In this study, a total of 1400 pregnant women aged 27.0 ± 4.5 years were randomly selected from the China Nutrition and Health Survey 2010–2012 (CNHS 2010–2012). The concentrations of 14 serum trace elements were determined by high-resolution inductively coupled plasma mass spectrometry. Reference values were calculated covering the central 95% reference intervals (P2.5–P97.5) after excluding outliers by Dixon’s test. The overall reference values of serum trace elements were 131.5 (55.8-265.0 μg/dL for iron (Fe), 195.5 (107.0–362.4) μg/dL for copper (Cu), 74.0 (51.8–111.3) μg/dL for zinc (Zn), 22.3 (14.0–62.0) μg/dL for rubidium (Rb), 72.2 (39.9–111.6) μg/L for selenium (Se), 45.9 (23.8-104.3) μg/L for strontium (Sr), 1.8 (1.2–3.6) μg/L for molybdenum (Mo), 2.4 (1.2–8.4) μg/L for manganese (Mn), 1.9 (0.6–9.0) ng/L for lead (Pb), 1.1 (0.3-5.6) ng/L for arsenic (As), 835.6 (219.8–4287.7) ng/L for chromium (Cr), 337.9 (57.0–1130.0) ng/L for cobalt (Co), 193.2 (23.6–2323.1) ng/L for vanadium (V), and 133.7 (72.1–595.1) ng/L for cadmium (Cd). Furthermore, some significant differences in serum trace element reference values were observed between different groupings of age intervals, residences, anthropometric status, and duration of pregnancy. We found that serum Fe, Zn, and Se concentrations significantly decreased, whereas serum Cu, Sr, and Co concentrations elevated progressively compared with reference values of 14 serum trace elements in pregnant Chinese women. The reference values of serum trace elements established could play a key role in the following nutritional status and health risk assessment.