This study suggested a new method to produce the in vitro cartilage tissues by cartilage differentiation in spheroids from adipose-derived stem cells on porous scaffolds. Adipose-derived stem cells (ADSCs) were used to produce spheroids by the hanging-drop method. The spheroids were then loaded into a porous scaffold and induced to differentiate into cartilage. To confirm the cartilage phenotype of the differentiated spheroids in the scaffold, the complex was evaluated for the expression of chondrogenic-related genes and proteins. The cartilagenous tissues formed from the spheroid-scaffold complexes were primarily checked for in vivo functioning by transplanting them into rat models of cartilage damage. The results showed that the spheroid-scaffold complexes displayed the cartilage phenotype after inducing chondrogenic differentiation. The complexes stained positive with safranin O, alcian blue and collagen 2, significantly expressed Sox9, Col2, and aggrecan genes compared with that before differentiation. In vivo, the differentiated spheroid-scaffold complexes formed mature cartilage that stained strongly positive with safranin O and fast red. These results suggest a promising strategy to produce cartilaginous microtissue for regenerative medicine.