The L858R mutation of epidermal growth factor receptor (EGFR) in nonsmall cell lung cancer is associated with the increased sensitivity to EGFR tyrosine kinase inhibitors. In this paper, a simple and sensitive method for identification of L858R mutation in cell lines and tumor tissues was developed using cationic conjugated polymer-based fluorescence resonance energy transfer technology (CCP-based FRET). The new detection system can detect even as low as 4-8% mutation of the total DNA. Through the detection results for 48 DNA samples from tumor tissues, a sensitivity of 95.24% (20/21) and a specificity of 96.30% (26/27) were demonstrated. Further, the application of this method in clinical molecular diagnosis was validated by detecting T790 M in EGFR of 35 patients. In comparison with DNA sequencing and real-time PCR methods, our new protocol simplifies procedures by eliminating the need for primer labeling, cumbersome workups and sophisticated instruments and improves sensitivity by amplifying fluorescence signals. Our CCP-based FRET technology is particularly attractive because of its higher sensitivity, cost-effective, and simple characteristics. Particularly, this new method could confirm the suspected positive samples arisen by DNA sequencing and real-time PCR methods. Thus, the CCP-based FRET technology opens up an avenue for clinical therapy by guiding medication to lung cancer patients responsive to anti-EGFR therapy.