The invention discloses an accurate and efficient eukaryotic biological gene identification method based on a transcriptome. The method comprises 1, quasi-identification gene expression prediction, 2,acquisition of a quasi-identification gene high expressed sample, RNA extraction and reverse transcription, 3, transcriptome sequencing, 4, transcriptome data analysis and storage, 5, transcriptome data retrieval and 6, gene identification primer design and gene identification. Through the close specie research report analysis, the transcriptome of the high expressed sample of the gene to be identified is determined, the target sequence of the gene to be identified is screened in the transcriptome, and the gene is identified and cloned based on the target sequence so that an accurate gene sequence is obtained. The method is suitable for identification of functional genes of most eukaryotes, is very accurate, satisfies the research demands of the functional gene identification in the research process, has a reasonable design, is easy to use, realizes a low cost and is suitable for promotion and use.