The adsorption behaviors and transfer pathways of antibiotics in plant–soil system are greatly influenced by the electrochemical properties of both soil particles and plant roots. However, the effects of roots electrochemical properties on antibiotic adsorption are largely unknown. Here, the fresh soybean, maize, and wheat roots with different electrochemical properties were obtained from hydroponic cultivation, and the adsorption processes and mechanisms of doxycycline, tetracycline, sulfadiazine, and norfloxacin on roots under various environmental conditions were investigated. Results showed that the adsorption amount of antibiotics on roots increased with the initial concentration of antibiotics. The coexisting low–molecular weight organic acids and anions inhibited the antibiotic adsorption on roots. The soybean roots performed strong adsorption ability compared with the maize and wheat roots driven by the variations in root electrochemical properties. This study demonstrates the significance of electrochemical interactions between antibiotics and roots in plant–soil system and can contribute to the more accurate risk assessment and effective pollution control of antibiotics.