Phenylalanine ammonia-lyase (PAL) gene plays an important role in the synthesis of flavones, lignin, and other bioactive compounds in living organisms. Inonotus bautnii, the only known flavone-producing filamentous fungus, is of great importance in the investigation of flavone metabolic pathways. To study the function of PAL enzyme in I. baumii flavone synthesis, a full-length cDNA of pal gene was cloned from I. baumii using DOP-PCR and RACE-PCR. The 2502-bp PAL coding region encodes an 833 amino acid protein with an approximate MW of 88.2 kDa. Three introns and four exons are present in the DNA sequence of IbPAL. Amino acid sequence alignment showed that IbPAL shares 76% similarity with PALs of Inonotus fungi. The three-dimensional structure of IbPAL showed that it is composed of an MIO domain, a core domain and an inserted shielding domain. On this basis, the IbPAL was expressed and purified using the prokaryotic expression vector pSMART-V with a 6xHis-tag in Escherichla colt, and its enzymatic activity was subsequently detected. Our results will aid in understanding the enzymatic properties of PAL and further confirm the mechanism of flavone synthesis in I. bawnii.