Abstract: Total RNA was isolated from walnut cotyledon by the methods of Trizol, Guanidine Thioeyanate, SDS and improved CTAB，according to walnut cotyledon characteristics of rich phenols, pigment, polysacchride and oil. The method of RNA isolation suitable for cotyledon of walnut was established through the rate of yield，purity and electrophoresis. The results showed that the purity of RNA isolated by Trizol and SDS reagent was lower than improved CTAB. RNA isolated by the Guanidine Thioeyanate dispersed in the pattern of small molecular. RNA isolated by the improved CTAB showed two clear bands28S rRNA, 18S rRNA respectively and the 28S was two times length as 18S, which means little RNA is decomposed by the method of improved CTAB reagent and the purity of RNA is very high. Yield rate of RNA extracted by the improved CTAB was (100±5.31） μg/g. Its ratio of A260/A280 was 1.98RTPCR amplification also showed that the quality of RNA isolated from the improved CTAB can meet the demands of molecular biology experiment.