Objective To explore the feasibility of fish collagen in constructing tissue-engineered cartilage. Methods Porous fish collagen was fabricated by lyophilization. Auricular chondrocytes derived from rabbit were seeded into the porous fish collagen. After cultured in vitro for 2 or 4 weeks, the cytocompatibility and the capacity of cartilage-specific extracellular matrix formation based on fish collagen were investigated. Thereafter, the cell-scaffold constructs were implanted into the rude mice subcutaneously. Gross observation, histological observation and quantitative evaluation of neocartilage test were carried out after cultured for 6 weeks. Results Fish collagen possessed satisfactory cytocompatibility, and facilitated cartilage-specific extracellular matrix formation both in vitro and in vivo. Notably, HE staining showed typical mature lacuna after cultured in vitro for 4 weeks or cultured in vivo for 6 weeks. After cultured in vivo for 6 weeks, Safranin-O staining showed abundant proteoglycan, biomechanical and biochemistry analyses indicated that neocartilage was comparable than native cartilage. Conclusion Fish collagen is a promising natural derived material as scaffold in constructing tissue engineering cartilage.