Atropine is a tropane alkaloid found in abundance in Datura plant. We used two liquid–liquid extraction methods and magnet solid-phase extraction to compare the amount of atropine in these two types of plants (Datura innoxia and Datura stramonium). The surface magnetic nanoparticle Fe3O4 correction with an amine and dextrin, and finally, magnetic solid-phase extraction Fe3O4@SiO2-NH2-dextrin (MNPs-dextrin), was prepared. We determined the effect of significant parameters in the removal step and optimization of atropine measurements with half-fractional factorial design (25–1) and response surface methodology via central composite design. The optimum conditions are for desorption solvent = 0.5 mL methanol and desorption time of 5 min. We obtained an extraction recovery of 87.63% with a relative standard deviation of 4.73% via six frequented measurements on a 1 μg L−1 atropine standard solution based on the optimum condition. Preconcentration factors for MNPs are 81, limit of detection = 0.76 μg L−1 and limit of quantitation = 2.5 μg L−1.