In order to provide resources of rice seedling substrate for mechanical transplanting, the effect of straw substrates with different preparation components and under different preparation processes on rice seedling growth was studied. The results showed that preparation components and preparation processes of straw substrates had significant effect on rice seedling quality. The emergence rates of treatment retted with fertilizer and fermentation bacteria and the treatment retted without fertilizer were 5%-10% higher than that of other treatments. The treatment retted with soil and fermentation bacteria increased rice seedling height, cauloid width, dry matter per plant, chlorophyll and nitrogen content, reduced plant sugar and nitrogen ratio, significantly improved the quality of rice seedling. Seedling soluble sugar content of treatment with soil was 1- 13 mg/g higher than that of straw substrate treatment, the starch content was 8-28 mg/g lower, and the ratio of sugar and nitrogen was lower than that of straw substrate treatment. The seedlings comprehensive quality of machine transplanting rice were relatively good with nitrogen content of 30 mg/g, non-structural carbohydrate content of 65-80 mg/g, sugar and nitrogen ratio of 2.5. Straw could replace nutrient soil as substrate in rice machine transplantation. Straw substrate retted with a little soil and fermentation bacteria had better effect.