Traditional approaches in tissue engineering are limited in that cell seeding is inefficient and cells cannot be located on a scaffold precisely. Moreover, the traditional methods, which rely on a random and probabilistic process, produce scaffolds with low regularity in porosity, pore size, and interconnection of pores. In this research, we propose a novel method to fabricate a scaffold for tissue engineering, which can overcome the limitations of traditional approaches. Cell-encapsulated alginate solution and cross-linker solution were laminarly flowed into a microfluidic channel. Then, the alginate solution was gelled to form a cell-encapsulated alginate microfiber by the diffusion of gelation ion from the cross-linker solution and ejected from the outlet of channel to the reservoir. The diameter of the fabricated microfiber can be controlled by the flow rate ratio of the two solutions. Moreover, this method, which has no cell seeding step, eliminates the possibility of loss of cells and the problems related to distribution of cells. We also show the feasibility of the alginate microfiber as a scaffold, which can promote chondrogenesis. The chondrogenesis in the alginate microfiber was evaluated by both histological and biochemical analyses. The increase of major markers of chondrogenesis such as glycosaminoglycan and collagen shows the potential of alginate microfiber as a scaffold for cartilage.