Plenty of studies have demonstrated that DIF has an effect on the fruit growth. To study the effects of day and night temperature differences on the strawberry sugar quality, an experiment using climate chambers was conducted. Five different differences between the day and night temperatures (DIF) were set, which were 6 °C (28 °C/22 °C, day/night temperature), 8 °C (29 °C/21 °C), 10 °C (30 °C/20 °C), 12 °C (31 °C/19 °C), 14 °C (32 °C/18 °C). The results showed the following indices peaked with a DIF of 12 °C, including the photosynthesis rate, glucose content, fructose content, sucrose content, soluble sugar content and sugar metabolic enzyme activity. The respiration rate increased with the DIF during the day and decreased with the DIF at night. The root dry weight peaked at a DIF of 10 °C, the stolon dry weight peaked at a DIF of 8 °C and the leaf dry weight peaked at a DIF of 6 °C; however, the fruit dry weight reached maximum values at a DIF of 12 °C. The Grey correlation analysis showed that the most important factor in our experiment affecting the fructose content was the sucrose phosphate synthase; however, for the sucrose, glucose, and soluble sugars, the most important factor was the photosynthesis. We found that a DIF of 12 °C (31 °C/19 °C, day/night temperature) was the most suitable for strawberry growth, especially for the sugar content accumulation.