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The effect of pre-thermal treatment on anatase TiO2 with high specific surface area (ca. 334 m2/g, HK) was investigated for dye-sensitized solar cell (DSC). The optimized substrate was observed on the HK pre-calcined at 450°C (HK-450) in terms of morphological feature, crystallinity, surface area, and photocurrent density. A DSC fabricated with HK-450 exhibited the high photovoltaic performance with significantly improved short-circuit current. This was attributed to the superior morphological features of HK-450, causing the high adsorption of dye and high photocurrent density. The optimization between the morphological feature, crystallinity, specific surface area and the photocurrent density of TiO2 substrate was found to give rise to the improved overall conversion efficiency of DSC.