Traditionally, the structural details of microbial cell walls are studied by thin-section electron microscopy, a technique that is very demanding and requires vacuum conditions, thus precluding live cell experiments. Here, we present a method integrating single-molecule atomic force microscopy (AFM) and protein design to measure cell wall thickness in a living yeast cell. The basic idea relies on the expression of His-tagged membrane sensors of increasing lengths in yeast and their subsequent specific detection at the cell surface using a modified AFM tip. After establishing the method on a wild-type strain, we demonstrate its potential by measuring changes in cell wall thickness within a few nanometers range, which result from (bio)chemical treatments or from mutations affecting the cell wall structure. The single molecular ruler method presented here not only avoids cell fixation artifacts but also provides new opportunities for studying the dynamics of microbial cell walls during growth, drug action, or enzymatic modification.