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Large-scale engineered constructs comprising precisely organized cellular microenvironments is of great importance for tissue engineering. This paper addresses an improved method for fabricating segmented magnetic hydrogel fiber based on microfluidic device for the scaffold application in tissue regeneration. The fabricated hydrogel fiber is made of alginic acid sodium and with a diameter of 34 μm. MRI contrast media solution that is a hydrophilic colloidal solution of superparamagnetic iron oxide coated with carboxydextran is added into the alginic acid sodium solution to append magnetic material inside fiber. The magnetic material inside the hydrogel fiber is regulated by the microfluidic chip and controlled as 5 mm of pure hydrogel with an interval of 500 μm of magnetic hydrogel. Two magnetic manipulation systems based on Maxwell coil and electromagnet are utilized to evaluate the magnetic response of the magnetic hydrogel fiber. Evaluation results show the magnetic hydrogel fiber can be patterned and aligned with the proposed system. The cultivation results of hydrogel fiber with C2C12 cells shows the potential for real applications of proposed method in tissue engineering.