Pure culture is important for taxonomic, physiological and various application studies of fungi. Many protocols for obtaining pure cultures have been reported, of which single spore isolation was used most frequently in mycological studies. However, previously reported methods were relatively complicated and time consuming. In this paper, an optimized protocol of single spore isolation is introduced. Compared to previous protocols, this protocol is favorable in terms of its lower contamination rate, is less time consuming, and requires only simplified procedures with common and easily accessible apparatus.