The purpose of this experiment is to prepare the efficient rice seedling substrate with agricultural organic waste of lees as the main raw material. First, wheat straws and mushroom residues were fermented and decomposed completely into substrate material, then vermiculite and perlite were added as the ingredients to produce various formula substrates for rice seedling experiment in order to secreen the optimal one. The results showed that the above raw materials could be used to prepare rice seedling substrates. The formula of 60% compost (lees + wheat straws) + 30% vermiculite + 10% perlite (Treatment T6) had a best comprehensive effect, the plant height, contents of nitrogen, phosphorus and potassium as well as root activity of T6 treatment were 13.94%, 12.68%, 24.62%, 5.77% and 15.78% higher than those of the commercial rice seedling substrate (Treatment T8), respectively. The above results provide theoretical bases for the resource utilization of lees and other agricultural wastes and for the development of environment-friendly seedling substrates.