[Background] Single cell culture of fungi is very important to study cell heterogeneity and cell growth characteristics. Therefore, it is necessary to establish a simple and convenient method for culturing and observing fungal single cells. [Objective] To establish a method for capturing and culturing the single cell of fungi based on microfluidic. At the same time, localization and real-time observation of single cell will be done. [Methods] L-edit was used to design the pattern and the plasma bonding was used to produce the microfluidic chip. Rhodotorula glutinis solution and Trichoderma reesei spore solution were injected by syringe pump to capture single cell. Trypan blue was used to determine the survival rate of yeast cells. Germination, growth and reproduction of yeast single cells and spore were observed under microscope. [Results] The microfluidic chip was intact and can be used in single cell capture of yeast or spore. The capture rate of the yeast was 25.00%±1.38%. The budding process of yeast cells was observed at 0, 2, 4 and 6 h. There was no significant difference between the survival rate of yeast cultured in chip and in shaking culture until 48 h. The process of spore germination and mycelium growth was observed at 0, 3, 6 and 9 h. Moreover, the mycelium kept growing until 120 h. [Conclusion] A microfluidic chip was designed and produced to capture, culture and localization the single cell of Fungi. This is the first application of this kind of chip in the fungal single cell culture. The cell can grow normally in this chip for at least 2 days, and 5 days even longer cell culture could be achieved by this way. The microfluidic chip can achieve intuitionistic localization and real-time observation of fungal single cell. It has the potential to study physiological and genetic characters of a variety of microorganism.