Objective: To establish reference values for the amniotic fluid index (AFI) measurement between 26w0d and 41w6d of gestation in a Brazilian population. Methods: We performed a cross-sectional study with 1984 low-risk singleton pregnant women between 26w0d and 41w6d of gestation. AFI was measured according to the technique proposed by Phelan et al. Maternal abdomen was divided into four quadrants using the umbilicus and linea nigra as landmarks. Single vertical pocket in each quadrant was measured and the AFI was generated by the sum of these four values without umbilical cord or fetal parts. All ultrasound exams were performed by only two experienced examiners. AFI was expressed as median, interquartile range, mean and ranges in each gestational age (GA) interval. Polynomial regressions were performed to obtain the best fit with adjustment by the determination coefficient (R2). Results: Mean of AFI ranged from 14.0 ± 4.1 cm (range, 9.7–14.0) at 26w0d to 8.3 ± 4.7 cm (range, 1.9–16.5) at 41w6d, respectively. The best polynomial regression fit curve was a first-degree: AFI = 16.29–0.125*GA (R2 = 0.01). According the scatterplot, AFI values practically did not vary with advancing GA. Conclusion: Reference values for the AFI measurement between 26w0d and 41w6d of gestation in a low-risk Brazilian population were established.