Corn stalk is a kind of newly substrate that can replace peat. It is important to study the application technique for horticultural crops cultivation and seedling production. The effect of the volumetric ratio of vermiculite to corn stalk substrate，the adding quantity of chicken manure and the relative moisture content on the growth of tomato plug seedling were studied by using orthogonal test design. The results showed that the important order of different factors to the above-ground growth of tomato seedlings was: the quantity of chicken manure > the relative moisture content > the volumetric ratio of vermiculite and chicken manure, while the order to the under-ground growth was: the moisture content > the volumetric ratio of vermiculite and corn stalk substrate > the quantity of chicken manure. The optimal combination was obtained in this experiment, which the relative moisture content was 85% and the volumetric ratio of vermiculite to Corn stalk substrate 1:1, and the quantity of chicken manure 15 g in per plug trays. The results of this experiment provided a useful reference to the utilization of corn stalk substrate and developing new type substrates.