The objective of this paper was to optimize the fermentation conditions of lactose-free yogurt. The level of residual sugar was used as response variable. Moreover, lactose-free yogurt and ordinary were prepared under the same conditions and their sensory characteristics were compared. Also, differences in their instrumental quality characteristics were assessed using an electronic eye, an electronic nose and an electronic tongue. The results showed that the optimal fermentation conditions were as follows: lactase dose 3.9 U/L, inoculum size 2 U/1 000 kg, fermentation temperature 43 ℃ and time 5.5 h. The lactose content of the yogurt prepared under the optimized conditions was (0.29 ± 0.07) g/100 mL. The lactose-free yogurt showed a 1.5-fold increase in lactic acid bacterial count than the ordinary yogurt (P < 0.05), but there was no significant difference in acidity or viscosity between them (P > 0.05). Principal component analysis (PCA) demonstrated no significant difference (P > 0.05) in color, aroma or taste. Therefore, compared with the ordinary yogurt, the lactose-free yogurt had no significant difference in sensory quality.