Unconjugated estriol (uE₃) is one of the main naturally occurring estrogens that plays an important role in growth and development of the fetus. Usually, the level of uE₃ is very low in men and non-pregnant women, but in pregnant women, the level of estriol has been found to be quite high. Therefore, the combination of uE₃, AFP, and hCG is now widely used for Down Syndrome screening as a triple marker. Here, we developed a superparamagnetic lateral flow immunochromatographic assay to quantitatively detect uE₃. The detection limit of this assay was 0.86 nmol/L and the linear range for the determination of uE₃ was from 1 to 100 nmol/L. The detection time was 15 min and the assay had very low cross-reactivity with estrone (E₁), estradiol (E₂), and progesterone. The coefficient of variation (CV) of intra- and inter-assay ranged from 5% to 13%. The magnetic signals were stable under 37 °C within 7 d. Moreover, the concentrations of uE₃ measured by lateral flow immunochromatographic assay in 230 serum samples collected from pregnant women at the Chinese People’s Liberation Army General Hospital had a good correlation with those measured by time-resolved fluorescence immunoassay (R = 0.946).