The fluorescent probe L, based on naphthalimide-modified coumarin, was designed, synthesized, and characterized, which could recognize Cu²⁺ from other cations selectively and sensitively in HEPES buffer (10 mM, Ph = 7. 4)/CH₃CN (1:4, V/V). When the probe L interacted with Cu²⁺, the color and the fluorescent intensity changed obviously and it provided the naked-eye detection for Cu²⁺. The recognition mode between them was achieved by Job's plot, IR, MS, SEM, and ¹HNMR. In addition, test strips made from L could still interact with Cu²⁺ in tap water effectively. The limit of detection (LOD) of L was 3.5 × 10⁻⁶ M. Additionally, the density functional theory (DFT) calculation method was used to analyze the action mechanism of L toward Cu²⁺. Importantly, the fluorescent probe L could demonstrate favorable selectivity toward Cu²⁺ in Caenorhabditis elegans. Thus, L was considered to have some potential for application in bioimaging.