A novel selective and reliable ratiometric fluorescence probe has been successfully synthesized for precise, sensitive, and simple quantitation of methotrexate (MTX). Hydrothermal method was employed to fabricate nitrogen-doped carbon dots using Annona squamosa seeds (AS-CDs) as a starting material, and their characteristics were confirmed using transmission electron microscopy (TEM), UV-Vis spectroscopy, fluorescence spectroscopy, X-ray diffractometry (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). The ratiometric fluorometric assay, which is based on measuring the ratio of emissions (F₃₅₅/F₄₃₀), has a wide detection range of 5–2000 ng /mL and a limit of detection (LOD, S/N = 3) of 1.5 ng /mL. The developed sensing method was successfully applied to the quantification of MTX in rabbit plasma samples and parenteral formulations, achieving satisfactory recoveries %. Magnetic molecularly imprinted solid-phase microextraction was used for selective extraction of MTX from plasma samples. The pharmacokinetic parameters were successfully determined in real rabbit plasma samples after intravenous administration of MTX. The as-designed probe does not only improve the sensitivity, but also enhances the precision and accuracy of the proposed method. Overall, this study presents a promising approach for the detection of MTX in genuine samples with acceptable degree of selectivity and sensitivity.