Abstract: In order to realize the resource utilization of waste resources, the pig-breeding litters from the microbial fermentation bed were used to develop the tomato seedling substrate. By selecting the pig-breeding litters used in the microbial fermentation bed for one year, its leaching liquor was extracted and diluted into different concentrations. The germination of tomato seeds was used as an indicator to screen the appropriate amount of litter in the substrate. Then, the basic substrate formula was determined by the plug seedling experiment, and the usage of N, P and K was further optimized. Finally, the tomato seedling substrate formula was determined. The results showed that the formula for the preparation of tomato seedling substrate by using the pig-breeding litters from the microbial fermentation bed was as follows: the ratio of litter to coconut husk was 3 : 7 (volume ratio), the dosage of N was 0.3 kg·m⁻³, the dosage of P was 0.2 kg·m⁻³, and the dosage of K was 0.1 kg·m⁻³. By using the prepared substrates for the tomato seedling, the emergence rate of seedlings could reach up to 98%, and the plant height, stem diameter and root length of the seedlings were all significantly better than those of the commercial peat substrate (the control group). After these seedlings were transplanted in the field, the plant height and bearing fruit number of tomato seedlings in the field were significantly better than those of the control group. The preparation of tomato seedling substrate could realize the resource utilization of pig-breeding litters from the microbial fermentation bed.