Uranium is one of the most common radioactive contaminants in the environment. As a major nuclear material in production, environmental samples (like soil and groundwater) can provide signatures on uranium production activity inside the facility. Thus, developing a new and portable analytical technology for uranium in aqueous media is significant not only for environmental monitoring, but also for nonproliferation. In this work, a label-free method for the detection of uranyl (UO₂²⁺) ions is developed by monitoring the translocation of a peptide probe in a nanopore. Based on the difference in the number of peptide events in the absence and presence of uranyl ions, nanomolar concentration of UO₂²⁺ ions could be detected in minutes. The method is highly selective; micromolar concentrations of Cd²⁺, Cu²⁺, Zn²⁺, Ni²⁺, Pb²⁺, Hg²⁺, Th⁴⁺, Mg²⁺, and Ca²⁺ would not interfere with the detection of UO₂²⁺ ions. In addition, simulated water samples were successfully analyzed.