This in vitro study aimed to accelerate the remineralization of a completely demineralized dentine collagen block in order to regenerate the dentinal microstructure of calcified collagen fibrils by a novel electric field-aided biomimetic mineralization system in the absence of non-collagenous proteins. Completely demineralized human dentine slices were prepared using ethylene diamine tetraacetic acid (EDTA) and treated with guanidine hydrochloride to extract the bound non-collagenous proteins. The completely demineralized dentine collagen blocks were then remineralized in a calcium chloride agarose hydrogel and a sodium hydrogen phosphate and fluoride agarose hydrogel. This process was accelerated by subjecting the hydrogels to electrophoresis at 20 mA for 4 and 12 h. X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM) were used to evaluate the resultant calcification of the dentin collagen matrix. SEM indicated that mineral particles were precipitated on the intertubular dentin collagen matrix; these densely packed crystals mimicked the structure of the original mineralized dentin. However, the dentinal tubules were not occluded by the mineral crystals. XRD and EDX both confirmed that the deposited crystals were fluorinated hydroxyapatite. TEM revealed the existence of intrafibrillar and interfibrillar mineralization of the collagen fibrils. A novel electric field-aided biomimetic mineralization system was successfully developed to remineralize a completely demineralized dentine collagen matrix in the absence of non-collagenous proteins. This study developed an accelerated biomimetic mineralization system which can be a potential protocol for the biomineralization of dentinal defects.