A total of 127 strains of endophytic fungi were isolated from roots, branches and leaves of Huperzia serrata. These strains were identified into 19 genera based on morphological characters and ribosomal DNA (rDNA) sequence analysis, there into Penicillium, Aspergillus and Podospora were dominant populations in H. serrata. From analysis results we found some endophytic fungi showed a certain degree of tissue preference. The isolation rate and colonization rate of stems were both larger than those of leaf and roots. After testing the acetylcholinesterase (AChE) inhibitory activity of these endophytic fungi, a total of 39 endophytic fungi belonging to 15 genera showed AChE inhibition. Eleven endophytic fungi showed potent AChE inhibition, 7 of which were isolated from leaf. The research not only provided theoretical basis for developing and utilizing the resources of endophytic fungi in H. serrata but also showed a new path for searching medicines resource which has AChE inhibitory activity.