Objective The method for detecting expression of human CDK14 gene with Real-time quantitative PCR was developed.Methods To establish a method for detecting expression of human CDK14 gene with Real-time quantitative PCR by designing and synthesis of the primers of CDK14 target gene andβ-Actin reference gene and extracting total RNA from different lung cancer cell lines.Then the specificity,detection range and repeatability of this method were evaluated.At last,the expression level of CDK14 gene in different cell lines,which were with or without siRNA interference,were carried out by using this method.Results The method for detecting expression of human CDK14 gene with Real-time quantitative PCR,which had good specificity,good repeatability (CV=7.3 ％) and wide detection range (Ct value range of CDK14 and β-Actin amplification curve were 22.47～32.96 and 15.14～ 27.55 respectively,r2 =0.9844),was developed and it was verified by electrophoresis analysis,melting curve,PCR product sequencing.And CDK14 gene expression level,which was detected by this method,increased in HCC827 D5,H1650 and number 1 siRNA segment was effective interference segment.Conclusion The method for detecting expression of human CDK14 gene with Real-time quantitauve PCR was established successfully.