An improved method was utilized for constructing hybrid arrested λ phage cDNA library by subtractive hybridization, in which magnetic particles were applied to separate monomers from hybrids, and candidate sscDNAs were enriched by subtracting two control mRNAs from one treated sscDNA. The results showed that this new approach had a efficient improvement in constructing cDNA library which increased the yield of monomers, simplified experimental procedures and enriched more candidate genes.