We present a simple and high-throughput microfluidic system for diffusion-based monolayer yeast cell culture monitoring. Yeast cells are patterned into the micro-cavity array with a suitable height (4 μm) that keeps the cells fixed in monolayer during the cell division. Different sizes of cavities and different repeating times of injection were tested in order to realize as many single-cell/cavity as possible. Single-cell/cavity has been achieved in about 40% of 100 parallel cavities. As a demonstration, we apply this technology to investigate budding yeast and fission yeast cultures and show that it permits single-cell resolution over many cellular generations. Our results show that the technique provides an easy way to study the phenotype of single yeast cell cycle or cell-cell communication in high-throughput microfluidic system.