Profound physiological changes during pregnancy may affect the requirement of retinol and tocopherol, which are essential micronutrients for the maintenance of maternal health and foetal development. However, the current reference intervals (RIs) of retinol and tocopherol are based on non-pregnant population. In the present study, a liquid chromatography-tandem mass spectrometry quantitation method for serum retinol and α-tocopherol was established and validated. In addition, we established trimester-specific RIs of retinol and α-tocopherol using the data from paired screening test for 31,301 outpatients who participated in the prenatal vitamins A/E evaluation program at our hospital using the Hoffmann method, which is a simple indirect RI estimation method that does not require the recruitment of healthy subjects. Further, to explore the associations between the levels of retinol and α-tocopherol and the parameters of complete blood count (CBC), the results of retinol, α-tocopherol, and CBC of 1,977 pregnant outpatients in the third trimester were analysed. The testing interval between the levels of vitamins and CBC was no more than 7 days. Although no significant changes were noticed in the levels of retinol, the α-tocopherol levels continuously increased with normal physiological changes throughout pregnancy. Lower retinol levels were associated with the higher incidence of anaemia, whereas higher levels of retinol and lower levels of α-tocopherol were associated with higher platelet count.