In order to explore the substitution substrate for rice seedling on upland fields, this paper uses spent mushroom substrate to study the physical and chemical properties of substrate, enzymic activity and number of tillers during the cultivation of rice seedling on upland fields. The results show that at the three stages of rice seedling cultivation (two-leaf stage, three-leaf stage, four-leaf stage), the content of organic matter and EC in spent mushroom substrate is higher than in the control soil, pH is within the range suitable for the growth of rice, and other nutrients (total nitrogen, total phosphorus, total potassium, available nitrogen, available phosphorus) are slightly different in different periods; except phosphatase, there are significant differences in urease, catalase and sucrase between spent mushroom substrate and the control soil; the number of tillers under spent mushroom substrate is larger than under the control.