Drop-on-demand (DOD) microdroplet formation and deposition play an important role in additive manufacturing, particularly in printing of three-dimensional (3D) in vitro biological models for pharmacological and pathological studies, for tissue engineering and regenerative medicine applications, and for building of cell-integrated microfluidic devices. In development of a DOD based microdroplet deposition process for 3D cell printing, the droplet formation, controlled on-demand deposition and at the single-cell level, and most importantly, maintaining the viability and functionality of the cells during and after the printing are all remaining to be challenged. This report presents our recent study on developing a novel DOD based microdroplet deposition process for 3D printing by utilization of an alternating viscous and inertial force jetting (AVIFJ) mechanism. The results include an analysis of droplet formation mechanism, the system configuration, and experimental study of the effects of process parameters on microdroplet formation. Sodium alginate solutions are used for microdroplet formation and deposition. Key process parameters include actuation signal waveforms, nozzle dimensional features, and solution viscosity. Sizes of formed microdroplets are examined by measuring the droplet diameter and velocity. Results show that by utilizing a nozzle at a 45 μm diameter, the size of the formed microdroplets is in the range of 52–72 μm in diameter and 0.4–2.0 m/s in jetting speed, respectively. Reproducibility of the system is also examined and the results show that the deviation of the formed microdroplet diameter and the droplet deposition accuracy is within 6% and 6.2 μm range, respectively. Experimental results demonstrate a high controllability and precision for the developed DOD microdroplet deposition system with a potential for precise cell printing.