Nanoporous anodic titanium dioxide (ATO) layers were formed via a three-step anodization process in glycerol-based electrolyte containing 0.38 wt.% NH4F and 1.79 wt.% H2O. The effects of applied potential (10–70 V), duration of the third step of anodization (15, 30 and 60 min) and the previous usage of the electrolyte on formation of ATO layers were examined at the anodizing temperature of 40 °C. It was found that at 10 V in both the fresh and used electrolytes, the porous layers are observed for short anodizing times and the compact oxide layers were formed when the time was extended to 60 min. On the other hand, the uneven oxide layers were obtained at 70 V. The structure and surface morphology analyses showed that for the fresh electrolyte the pore diameter increases with increasing anodizing potential and time. However, for the used electrolyte the pore diameter increases with potential and decreases or remains constant with time. The latter one is attributed to the enhanced precipitation of hydrous titanium dioxide on the ATO surface. The precipitation of hydrous titanium dioxide is also confirmed by oxide thickness analyses performed for the fresh and used electrolytes. The current density-time plots showed that usage of the previously used electrolyte leads to retardation of the oxide formation as a result of partial depletion of the fluoride ions from the electrolyte. These results were confirmed by measurements of the electrolyte conductance. The calculated efficiency of the anodization process, based on the thickness of the oxide layers, are significantly higher for the fresh electrolyte than for the used one.