Limbal stem cell deficiency (LSCD) causes vision loss and is often treated by simple corneal epithelial cell transplantation with poor long-term efficiency. Here, we present a biomimetic bilayer limbal implant using digital light processing 3D printing technology with gelatin methacrylate (GelMA) and poly (ethylene glycol) diacrylate (PEGDA) bioinks containing corneal epithelial cells (CECs) and corneal stromal stem cells (CSSCs), which can transplant CECs and improve the limbal niche simultaneously. The GelMA/PEGDA hydrogel possessed robust mechanical properties to support surgical transplantation and had good transparency, suitable swelling and degradation rate as a corneal implant. Encapsulated CECs and CSSCs maintained viability and proliferative activity in the bilayer limbal implant. In vivo, both CEC-loaded and CEC/CSSC-loaded hydrogel could repair the corneal surface in the LSCD model effectively. Notably, the corneal epithelial healing was faster, and corneal opacity and neovascularization were minimal in CEC/CSSC-loaded group. These findings highlight the feasibility of 3D printing in limbal construction, providing CEC/CSSC-loaded limbal implants as a treatment strategy for LSCD and corneal blindness. STATEMENT OF SIGNIFICANCE: This study aimed to enhance the long-term prognosis of limbal epithelial cell transplantation in patients with limbal stem cell deficiency by developing a 3D limbal implant that encapsulates corneal epithelial cells and limbal niche cells simultaneously. The 3D printed implant offers the advantages of mimicking the natural layered limbal structure and were found to enhance the regenerative capacity of corneal epithelial cells, suppress inflammation, and alleviate corneal scarring in vivo. This study highlights the importance of limbal microenvironment remodeling in the treatment of limbal stem cell deficiency and the potential of 3D printing in the treatment of corneal diseases.Copyright © 2025. Published by Elsevier Inc.