A method for detecting a gene alteration in a biological sample, comprising steps of: (a) mixing a target RNA obtained from the biological sample, a template-switching oligo, a reverse transcriptase, and a reverse transcription primer in a mixture, wherein the template switch oligo comprises a first universal primer sequence and the RT primer comprises a second universal primer sequence; (b) subjecting the mixture to a condition under which reverse transcription occurs to provide a target DNA complementary to the target RNA, (c) amplifying the target DNA with a first pair of primers to obtain a first PCR product, wherein the first pair of primers comprise a gene specific primer and a universal primer, the gene specific primer comprises a 5'-end first or second adapter sequence and a 3'-end sequence that hybridizes to a target gene under stringent conditions, the universal primer comprises a 5'-end first or second adapter sequence and a 3'-end sequence comprising the first or second universal primer sequence, and the first and second adapter sequences are different from each other; (d) amplifying the first PCR product with a second pair of primers to obtain a second PCR product, wherein each of the second pair of primers comprises a 5'-end third or fourth adapter sequence and a 3'-end sequence that hybridizes to the first or second adapter sequence, respectively, and the third and fourth adapter sequences are different from each other; and (e) analyzing the second PCR product to detect the presence of the gene alteration, preferably, the second PCR product is analyzed by sequencing analysis utilizing at least one adapter sequence. (a) mixing a target RNA obtained from the biological sample, a template-switching oligo, a reverse transcriptase, and a reverse transcription primer in a mixture, wherein the template switch oligo comprises a first universal primer sequence and the RT primer comprises a second universal primer sequence; (b) subjecting the mixture to a condition under which reverse transcription occurs to provide a target DNA complementary to the target RNA, (c) amplifying the target DNA with a first pair of primers to obtain a first PCR product, wherein the first pair of primers comprise a gene specific primer and a universal primer, the gene specific primer comprises a 5'-end first or second adapter sequence and a 3'-end sequence that hybridizes to a target gene under stringent conditions, the universal primer comprises a 5'-end first or second adapter sequence and a 3'-end sequence comprising the first or second universal primer sequence, and the first and second adapter sequences are different from each other; (d) amplifying the first PCR product with a second pair of primers to obtain a second PCR product, wherein each of the second pair of primers comprises a 5'-end third or fourth adapter sequence and a 3'-end sequence that hybridizes to the first or second adapter sequence, respectively, and the third and fourth adapter sequences are different from each other; and (e) analyzing the second PCR product to detect the presence of the gene alteration, preferably, the second PCR product is analyzed by sequencing analysis utilizing at least one adapter sequence.