The invention discloses primers for amplification of an endophytic fungi ITS gene in plant tissue. The primers include a nested PCR first round amplification primer pair (a forward primer NSA3, and a reverse primer NLC2) and a nested PCR second round amplification primer pair. The invention also provides a method for amplification of the endophytic fungi ITS gene in plant tissue by the primers. The method consists of: extraction of a plant tissue genome and PCR amplification on the extracted template DNA, with the amplification including nested PCR first round amplification and nested PCR second round amplification. The method can acquire ITS gene fragments of fungi in plant tissue with high quality, and lays the foundation for accurate analysis of the biological diversity of plant endophytic fungi.