Phthalic acid esters (PAEs) are a group of organic compounds that show vulnerability effects in different stages of human development. In this work, two sensitive and efficient impedimetric biosensors (IBs) were introduced and their interactions with four PAEs, namely dibutyl phthalate (DBP), dimethyl phthalate (DMP), di(2-ethylhexyl) phthalate (DEHP), and dicyclohexyl phthalate (DCHP), in aqueous solutions with these biosensors were separately investigated via electrochemical impedance spectroscopy (EIS). The surface of a copper electrode was modified by azolla fern dried powder (AZ) and magnetite-modified azolla nanocomposites (MAZ NCs) to form an azolla-based impedimetric biosensor (AZIB) and magnetite azolla nanocomposite-based impedimetric nanobiosensor (MAZIB), respectively. Determinations of PAEs with the designed biosensors were conducted based on their blocking effect on the biosensor surface to ferrous ions oxidation. After each impedimetric measurement, the electrode surface was covered again with the modifier. Nyquist plots were obtained and indicated that the charge-transfer resistance (R_CT) values of the bare electrode, AZIB, and MAZIB without injection of PAEs were 468.8, 438.7, and 285.1 kΩ, respectively. After the separate injection of DBP, DMP, DEHP, and DCHP (3 μg L⁻¹) on the surface of AZIB and MAZIB, R_CT values were obtained as 563.9, 588.5, 548.7, and 570.1 kΩ for AZIB and 878.2, 1219.2, 754.3, and 814.7 kΩ for MAZIB, respectively. It was observed that the PAE blockers with a smaller structure provided better point-by-point coverage of the surface, which led to a bigger shift in R_CT. The linear relationship between the EIS responses and each PAE concentration was investigated in the range of 0.1–1000 μg L⁻¹. The limit of detection (LOD) and limit of quantification (LOQ) values were obtained in the ranges of 0.003–0.005 μg L⁻¹ and 0.010–0.016 μg L⁻¹ for AZIB and 0.008–0.009 μg L⁻¹ and 0.027–0.031 μg L⁻¹ for MAZIB, respectively. The results showed that these biosensors can be used to determine PAEs in real aqueous samples with good relative recoveries ranging from 93.0–97.7% (RSD < 2.58%) for AZIB and 93.3–99.3% (RSD < 2.45%) for MAZIB. The results confirmed that these impedimetric biosensors offer high sensitivity and performance for the determination of trace PAEs in aqueous samples.