The orientation of the organofibrous structure plays an important role not only in the biomineralization process but also in the extracellular matrix (ECM) of articular cartilage by providing cells with biomechanical cues. Here, it is reported that a long range nanofibrous orientation can be realized by a self-assembling ionic complex between (+)-charged amphiphilic peptide block copolymers with a rigid-flexible block structure (polyalanine-PLX-polyalanine; PA-PLX-PA) and (-)-charged hyaluronic acid (HA). A biomimetic 3D culture system encapsulating chondrocytes is formed by a temperature-sensitive sol-to-gel transition of the PA-PLX-PA/HA complex aqueous solution, which provides a compatible microenvironment for the cells. The cell proliferation and biomarker expression for articular cartilage are significantly improved in the PA-PLX-PA/HA complex system relative to the PA-PLX-PA or the commercially available Matrigel systems. In particular, noticeable cell clustering is observed in the PA-PLX-PA/HA complex system with the long range nanofibrous structure. This research suggests a new method for developing a nanofibrous structure using an amphiphilic peptide block copolymer and demonstrates its potential uses as a unique biomimetic cell-culture matrix.