3D printing of biological architectures that mimic the structural and functional features of in vivo tissues is of great interest in tissue engineering and the development of transplantable organ constructs. Printable bio-inks that are compatible with cellular activities play critical roles in the process of 3D bio-printing. Although a variety of hydrogels have been used as bio-inks for 3D bio-printing, they inherit poor mechanical properties and/or the lack of essential protein components that compromise their performance. Here, a hybrid Matrigel–agarose hydrogel system has been demonstrated that possesses both desired rheological properties for bio-printing and biocompatibility for long-term (11 days) cell culture. The agarose component in the hybrid hydrogel system enables the maintenance of 3D-printed structures, whereas Matrigel provides essential microenvironments for cell growth. When human intestinal epithelial HCT116 cells are encapsulated in the printed Matrigel–agarose constructs, high cell viability and proper cell spreading morphology are observed. Given that Matrigel is used extensively for 3D cell culturing, the developed 3D-printable Matrigel–agarose system will open a new way to construct Matrigel-based 3D constructs for cell culture and tissue engineering.