The invention provides an in-vitro cell disease model and a preparation method thereof, and a drug screening method. The method for preparing the in-vitro cell disease model comprises the following step: carrying out linear dissection on a preset position of cells by using a laser microdissection system so as to obtain the in-vitro cell disease model. The method for preparing the in-vitro cell disease model can be used for preparing specific cell structures and or even a single cell disease model without damage to the non-model making region. The method is simple to operate; and the obtained in-vitro cell disease model can truly and comprehensively reflect the disease pathogenesis or process.