A selection of aptamers specific for di(2-ethylhexyl) phthalate (DEHP) and development of electrochemical impedance spectroscopy (EIS) aptasensor are described in this paper. The aptamers were selected from an immobilized ssDNA library using the systematic evolution of ligands by exponential enrichment (SELEX). The enrichment was monitored using real-time quantitative PCR (Q-PCR), and the aptamers were identified by high-throughput sequencing (HTS), gold nanoparticles (AuNPs) colorimetric assay, and localized surface plasmon resonance (LSPR). The EIS aptasensor was developed to detect DEHP in water samples. After eight rounds of enrichment, HTS, AuNPs colorimetric assay, and LSPR analysis indicated that four aptamers had higher binding activity, and aptamer 31 had the highest affinity (Kd = 2.26 ± 0.06 nM). The EIS aptasensor had a limit of detection (LOD) of 0.103 pg/mL with no cross-reactivity to DEHP analogs and a mean recovery of 76.07% to 141.32% for detection of DEHP in water samples. This aptamer is novel with the highest affinity and sensitivity.