An effective and high-throughput method is demonstrated to produce a colloidal crystal with impressive crystallinity. The fabrication procedure involves a two-stage electrophoresis process in which a vertical electrophoretic deposition of polystyrene microspheres (495 nm) is conducted on an ITO substrate to form a colloidal crystal in size of 4 × 4 cm2, followed by a horizontal electrophoresis to improve the packing crystallinity. Relevant parameters associated with the horizontal electrophoresis step including voltage polarity, bipolar voltage offset, and operation frequency are investigated. Their effects over the crystallinity of the colloidal crystals are verified quantitatively and qualitatively by structural observation via scanning electron microscope and optical reflectance spectra. We determine that the imposition of an alternately-changing bias of 80/0/-120/0 V at 10 Hz (25 ms in each bias) enables the production of colloidal crystals with the largest grain size (106 μm) and the smallest grain boundary (170 nm), as compared to samples prepared from a single vertical electrophoresis step. We attribute the improved crystallinity of the colloidal crystal to the bipolar bias that engenders localized movements of the microspheres, rendering them to pack more closely with reduced defects.