Cell culture has become an indispensable tool to uncover fundamental biophysical and biomolecular mechanisms of cells assembling into tissues. An important advancement in cell culture techniques was the introduction of three-dimensional (3D) culture systems. In this study, the mutual fusion of chondrocyte pellets was promoted in order to produce large-sized tissue-engineered cartilage by a multiplexed 3D hanging drop culture and agarose mold method to optimize the means of cultivation. Cell proliferation, aggregation, cell morphology maintenance as well as cartilage related gene expression and matrix secretion in vitro and subcutaneous implantation models were evaluated. These results indicated that the multiplexed 3D hanging drop culture involving the fusion of small pellets into a large structure enabled the efficient production of 3D tissue engineered cartilage that was closer to physiological cartilage tissue in comparison to that of the agarose mold method.