Two-dimensional (2D) atomically thin quantum dots (QDs) possess extraordinary electrical and optical properties. However, fabricating high quality 2D QDs via a universal and reliable technique remains a challenge. Here, we report a simple strategy to prepare high quality, monolayer single crystal 2D QDs via ultrathin cutting 2D bulk single crystals by ultramicrotome, followed by an exfoliation process. The as-prepared 2D QDs have pristine surface, high quality, high monolayer yield and high photoluminescence quantum yield (the highest photoluminescence quantum yield of WS2 is 18%), which can be used as promising, low toxic, biocompatible, and good cell-permeability fluorescent labeling agents for in vitro imaging.