Provided are a nucleotide sequence, and a method for constructing an RNA target area sequencing library and an application thereof. The nucleotide sequence comprises a first general sequencing sequence located at terminus 5' and a random primer sequence located at terminus 3' which are connected to each other. The random primer sequence contains 6-10 random nucleotides. The sequence is used to perform random reverse transcription on RNA to obtain a first chain of cDNA, and then the first general sequencing sequence on the nucleotide sequence and upstream and downstream specific primers are used to perform two-step nested PCR on the first chain of cDNA obtained after the reverse transcription, to obtain a high-throughput targeted sequencing library.
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