Constructing an artificial cell wall (AFCW) based on the layer-by-layer assembly of polymer films to protect probiotics in harsh conditions is highly desirable. Early findings showed that encapsulating yeast cells by an AFCW improved the cell viability by 50% in antibiotic solution. However, the detailed molecular interaction mechanism remains unclear by experiments. Herein, two ciprofloxacin (CPFX) permeation models, including models 1 and 2 that were, respectively, composed of just the yeast cell membrane and the AFCW coating cell membrane, were investigated by molecular dynamics simulations. The free energy profiles delineating the permeation process of CPFX reveal that the permeation of CPFX through the cell membrane of model 2 is more difficult than through that of model 1. The analysis results show that the AFCW leads to two sharp increases in free energy barriers, amounting to 8.9 and 6.2 kcal/mol, thereby reducing the penetrating rate of CPFX into the cell membrane. Moreover, decomposition of the potentials of mean force into free energy components suggested that the electrostatic interactions of CPFX with the AFCW predominantly contributed to the high free energy barriers. The current results provide a good understanding of the protective mechanism of the self-assembled cell walls against CPFX and help to design other AFCWs.