Severe lacrimal duct defects, including congenital absence and traumatic obliteration, remain formidable clinical challenges. Current therapeutic strategies, such as Jones tube prosthesis implantation, are severely limited by poor biocompatibility, susceptibility to displacement, and retrograde infections caused by fluid reflux. To address these limitations, we developed a novel anti-reflux artificial lacrimal duct using three-dimensional (3D) printing that integrates biomimetic hydrodynamic design with a biochemically optimized microenvironment. A meticulously engineered photopolymerizable composite hydrogel—comprising 15% polyethylene glycol diacrylate (PEGDA), 3% acrylamide, and 5% gelatin methacryloyl (GelMA) (60% degree of substitution)—was formulated as a high-strength hydrogel ink. Using 3D printing, we precisely fabricated an artificial lacrimal duct with a biomimetic unidirectional valve that simulates the natural Hasner valve. Guided by proteomic screening, the luminal surface of the construct was subsequently functionalized with a young donor-derived decellularized extracellular matrix (dECM) coating, onto which epidermal stem cells (ESCs) were seeded. The structural PEGDA/polyacrylamide network endowed the construct with superior printability and the requisite mechanical resilience to withstand valve fatigue. Hydrodynamic characterizations demonstrated that the 3D-printed valve effectively prevented fluid reflux while maintaining normal drainage, providing a robust physical barrier against retrograde infections. Biologically, the incorporation of GelMA-60 into the bulk scaffold and the young dECM coating at the luminal interface established a rejuvenated biochemical niche that exhibited excellent biocompatibility and successfully induced the targeted differentiation of ESCs into the lacrimal epithelial lineage. By synergizing physical anti-reflux hydrodynamics with omics-driven microenvironment rejuvenation, this composite 3D-printed construct enables superior tissue integration and infection resistance, presenting a highly promising tissue-engineered alternative for lacrimal system reconstruction.