Single spore isolation is a fundamental approach in plant pathology and mycology to isolate and identify plant fungal pathogens from diseased samples.  However, routine single spore isolation procedure is time-consuming and has a high risk of contamination by other microorganisms.  In this study, we developed a rapid approach for isolating a single spore of the fungal pathogen, Pyricularia oryzae, from rice blast diseased leaves in the paddy field with low potential of contamination.  First, rice blast leaves with single lesions were selected in the paddy field, and a single lesion was cut out and pressed and dragged gently across the surface of water agar.  Next, a germinated single spore with a barely visible piece of agar was cut out of water agar with a dissecting needle under a stereomicroscope at approximately 120-fold magnification.  Last, the germinated single spore with water agar was transferred onto oatmeal tomato agar for growth and preservation.  Based on our experience, a skilled technician or student can successfully isolate single spore from over 150 independent diseased samples with nearly no contaminations in a single working day.  This approach is also suitable for isolating single spore from other fungal disease samples that produce abundant aerial conidia.