To develop a kind of extensive, cheap, and good quality substrate for potato micro-tubers breeding, quadratic-orthogonal-rotation-combination design was adopted to optimize the ratio of three substrates (mushroom residues, sand and perlite). From the regression model established between yield of micro-tubers (Y) and mushroom residue (X1), sand (X2)and perlite (X3), which was expressed as Y=36.331 63–1.819 65X1+3.391 04X2+2.568 47X3– 1.786 37X12–4.208 21X32+2.122 50X1X2+4.327 50X1X3, it showed that the yield of potato micro-tubers decreased in the order of perlite, mushroom residues, and sand. It was also concluded from the model that there was no significant interaction between sand and perlite to the yield, while the interaction between mushroom residues and sand become obvious, the same was true for mushroom residues and perlite. The volume ratio of mushroom residue, sand and perlite was optimized as follows: (1.677–2.000): (1.157–1.452): (1.000–1.260) according to multiple regression and frequency analysis.