The monitoring of sulfadiazine (SDZ) is of great significance for food safety, environmental protection, and human health. In this study, a fluorescent aptasensor based on MnO2and FAM-labeled SDZ aptamer (FAM-SDZ30-1) was developed for the sensitive and selective detection of SDZ in food and environmental samples. MnO2nanosheets adsorbed rapidly to the aptamer through its electrostatic interaction with the base, providing the basis for an ultrasensitive SDZ detection. Molecular dynamics was used to explain the combination of SMZ1S and SMZ. This fluorescent aptasensor exhibited high sensitivity and selectivity with a limit of detection of 3.25 ng/mL and a linear range of 5–40 ng/mL. The recoveries ranged from 87.19% to 109.26% and the coefficients of variation ranged from 3.13% to 13.14%. In addition, the results of the aptasensor showed an excellent correlation with high-performance liquid chromatography (HPLC). Therefore, this aptasensor based on MnO2is a potentially useful methodology for highly sensitive and selective detection of SDZ in foods and environments.