Vaccination against the hepatitis A virus (HAV) administered in two doses has been used effectively in universal child immunization programs in several countries. A single-dose vaccination was adopted in some low-income countries in an attempt to reduce costs without losing effectiveness. In 2014, single-dose universal vaccination was introduced in Brazil for children aged two years. Since such strategy is still not universally accepted, its efficacy should be compared to the two-dose strategy. To assess the humoral response after the single-dose HAV vaccination schedule, a cross-sectional study was conducted in Primavera do Leste, in Mato Grosso state, Central Brazil, including 265 children vaccinated through the National Immunization Program. Blood was collected by using a digital puncture and further applied to filter paper cards. Anti-HAV was detected in 218 out of 265 dried blood spots (DBS). Blood venous samples were collected from 34 out of 47 children who were not anti-HAV positive in DBS samples. Eighteen of them tested positive for anti-HAV, giving a final score of 93.6% (236/252) of seropositivity. In conclusion, this study demonstrated a high rate of anti-HAV positivity in the short term after single-dose hepatitis A vaccination in the population investigated. Moreover, the DBS was shown to be a reliable tool for detecting anti-HAV antibodies.