The bacterial contamination of dental unit waterline (DUWL) output water has been reported in many areas, posing a potential risk to patients and dental healthcare staff. As a common method for addressing bacterial contamination, it is still a challenge for the disinfecting solution to provide enduring antibacterial activity. Herein, an approach to developing an intelligent antibacterial hydrogel coating with a dual-layer structure on polyurethane (PU) is reported in this study to control bacterial contamination on the inner surfaces of the DUWL tubing. The antifouling lower layer, derived from sulfobetaine methacrylate and 2-hydroxyethyl methacrylate, and a bactericidal upper layer consisting of poly-L-lysine (PLys) were sequentially grafted on the surface of PU. The antifouling and bactericidal layers were connected by the Schiff base structure. The results of scanning electron microscopy indicated the successful synthesis of the hydrogel coating with the thickness of 30.7 μm. Peaks at 399.85 eV (N 1s) in X-ray photoelectron spectroscopy spectra and 1653 cm −1 in Fourier transform infrared spectra represented the formation of Schiff base structure. Besides, the hydrophilicity assay found a decrease in water contact angle from 98.471 ± 3.208°to 54.594 ± 1.156°, indicating the great hydrophilicity of the hydrogel coating. In vitro antibacterial experiments revealed that there were a few dead bacteria adhered to the surface of the hydrogel coating. When treated with an acidic solution, most of the dead bacteria were released. Following the regrafting of PLys, only a small amount of dead bacteria were observed on the surface of the hydrogel coating. It was found that the hydrogel coating showed antifouling− killing−releasing functions as well as regeneration properties. Moreover, the hydrogel coating exhibited great stability and favorable biocompatibility. Thus, the hydrogel coating with triple functions (antifouling−killing−releasing) is capable of constructing an intelligent PU surface and has potential for reducing bacterial adhesion to DUWL tubing in clinical applications.