Objective To construct a lambda phage cDNA library of human lung cancer cells A549λ，and to lay a foundation for screening for lung cancer therapeutic drugs and study on the molecular mechanism of action of the drugs. Methods Total RNA of human lung cancer cells A549λ was extracted and digested with DNase I，and switching mechanism at 5'end RNA transcription（SMART）technology was used to synthesize double-stranded cD⁃NA（ds-cDNA）. The short fragments of ds-cDNA were removed via gel extraction，and the product was digested with proteinase K and SfiI successively and ligated into enzymatically digested λTriplEx2 vector，followed by in vi⁃tro packaging to obtain the primary library. The primary library was then amplified and titrated，and polymerase chain reaction was used to identify the inserted fragments. Results The titer of the constructed primary library and amplified library of human lung cancer cells A549λ was 1.88×107 pfu/mL and 2.50×109 pfu/mL，respectively. The inserted fragments of cDNA ranged from 500 bp to 3 000 bp. Conclusion A lambda phage cDNA library of human lung cancer cells A549λ was successfully constructed，which lays the groundwork for screening for potential drugs targeted at lung cancer and study on the molecular mechanism of action of the drugs.