Cell-laden hydrogel fibers are widely used as the fundamental building blocks to fabricate more complex functional three-dimensional (3D) structures that could mimic biological tissues. The control on the diameter of the hydrogel fibers is important so as to precisely construct structures in the above 3D bio-fabrication. In this paper, a pneumatic-actuated micro-extrusion system is developed to produce hydrogel fibers based on the crosslinking behavior of sodium alginate with calcium ions. Excellent uniformity has been obtained in the diameters of the fabricated hydrogel fibers as a proportional-integral-derivative (PID) control algorithm is applied on the driving pressure control. More importantly, a linear relationship has been obtained between the diameter of hydrogel fiber and the driving pressure. With the help of the identified linear model, we can precisely control the diameter of the hydrogel fiber via the control of the driving pressure. The differences between the measured and designed diameters are within ±2.5%. Finally, the influence of the calcium ions on the viability of the encapsulated cells is also investigated by immersing the cell-laden hydrogel fibers into the CaCl₂ bath for different periods of time. LIVE/DEAD assays show that there is little difference among the cell viabilities in each sample. Therefore, the calcium ions utilized in the fabrication process have no impact on the cells encapsulated in the hydrogel fiber. Experimental results also show that the cell viability is 83 ± 2% for each sample after 24 h of culturing.