The invention relates to the field of molecular biology, and aims to provide a lily bulb total RNA extraction method suitable for Iso-Sep. The method includes the steps that a tuber tissue culture bulb is ground into powder and added into a lysate CLB and beta-mercaptoethanol mixed solution, and splitting is carried out until the mixture is in a homogenate state; the RNA concentration is increasedfrom the total amount by increasing the sample quantity and the volume of the lystate, beta-mercaptoethanol is added to be bonded with the lysate, and a series of operations are conducted, so that the splitting process is optimized, polysaccharides and phenols are effectively removed, and RNA can be better dissolved out from tissue; three-fold genome clearance columns are used for removing DNA; finally, the RNA is concentrated through an adsorption column so as to further improve the RNA concentration. According to the method, no toxic irritant reagents such as phenol and chloroform need to be used, so that the safety of operators is better protected; the operation is simple, the consumed time is short, the extracted RNA is good in integrity and high in purity, and the requirements of most molecular biological experiments such as full-length transcriptome sequencing can be met.