646 positive clones were randomly selected from the library of suppression subtractive hybridization (SSH) constructed at early stage. These clones were subject to sequencing and bioinformatics analysis. The results show that 639 high quality EST were got by sequencing, 186 unigenes was obtained, of which 89 contigs and 97 singletons were contained. Blast alignment with Nr database showed that 166 unigenes were of high similarity. KOBAS suggested that that glyoxylate and dicarboxylate metabolism, carbon metabolism, carbon fixation in photosynthetic organisms, biosynthesis of amino acids, nitrogen metabolism et al. played a key role in drought-resistant of rapeseed according to P-value. In the drought stress response, the genes of MYB family transcription factor, NAC5, zinc finger protein and other genes related to photosynthesis including malate dehydrogenase, ribulose 1,5-bisphosphate carboxylase/oxygenase, fructose 1,6-bisphosphate aldolase, phosphoribulokinase, carbonic anhydrase, aconitase like protein and delta 1-pyrroline-5-carboxylate synthetase A, F-box protein etc were inovled. The analysis of unigenes showed that a large share of function could be attributed to binding and catalytic activity. The high drought-tolerant rapeseed Holiday as experimental materials, were treated with drought stress during the flowering stage and with normal irrigation as the control group. We collected the samples at the 1st, 3rd, 5th and 7th day of drought stress(the soil water content was 60%, 40%, 30% and 20% respectively) and analyzed their expression values of P5CSb, BnSOS2 and CAM genes from screening the library by quantitative real time PCR reaction, their expression level were found up-regulated under drought stress in the leaves. This result suggested those genes may play an important role in the resistance of rape to drought stress.