Introduction: Nowadays, a major limitation for organoids culturing is the dependence on Matrigel. Matrigel's undefined makeup and batch variations as well as its animal source limit its use in human therapies. Consequently, researchers were exploring hydrogels with clearer compositions as replacements. Recent work has used programmable DNA-based hydrogels to grow organoids. However, integrating functional DNA aptamers into hydrogels for organoid culture hasn't been reported. This study aimed to develop a hydrogel made from cellulose and equipped with aptamers to culture cholangiocyte organoids (COs). Methods: EpCAM-CELAPT MINI dual-aptamer chains were synthesized by rolling circle amplification (RCA), which were conjugating with Nanocellulose fibrils (CNF) via CaCl2 to fabricate Aptamer-functionalized CNF (Apt/CNF) hydrogels. Material properties were characterized by scanning electron microscope (SEM), X-Ray Diffraction (XRD), Fourier transform infrared (FT-IR), and rheological testing. COs were derived from human bile duct tissues. Organoids grown in Apt-CNF hydrogel versus Matrigel controls were evaluated for: (1) viability (Calcein-AM/PI), (2) marker expression (PCR/immunofluorescence for EpCAM/CK19/CK18), and (3) function (ELISA for albumin/bile acid secretion). Results: Firstly, the SEM micrograph revealed a porous structure of hydrogel that facilitated cell adhesion and growth. The XRD analysis and the FTIR spectrum confirmed the presence of phosphorus, which indicated successful DNA incorporation within the material. Subsequently, by conducting the rheological detection we could easily find that the mechanical stability of Apt-CNF hydrogels was significantly higher than Matrigel which means it will remain stable for longer and more suitable for formation and proliferation of organoids. Finally, we used those two matrices to culture COs. The high expression of common biliary biomarkers of COs, such as cytokeratin 18, cytokeratin 19 and so on both on mRNA and protein levels demonstrated the well-organized establishment of COs. Unlike the compulsory hypothermia condition of Matrigel upon using, the Apt-CNF hydrogel could form easily at room temperature with the supplement of CaCl2. Also, it only took less than 15 minutes to solidify for Apt-CNF hydrogels compared to the 30 minutes of Matrigel. According to the Calcein-AM/PI staining, after 1, 3, 5 days of culturing, the organoid size and viability of COs of Apt-CNF hydrogels were equal to that of Matrigel while better than hydrogels without crosslinking with aptamer. Conclusion: The Apt-CNF hydrogels provide a chemically defined, human-compatible alternative to Matrigel. It allows precise control of COs growth with distinct benefits: rapid room-temperature gelation, liver-mimetic viscoelasticity, and lower costs. It better simulates the in vivo environment for disease modeling and holds strong potential for developing transplantable cholangiocyte organoids.