Abstract: In order to develop three-dimensional cell scaffolds for in vitro toxicological evaluation of cigarette smoke, a high internal phase emulsion method was applied to prepare the scaffolding material. Human lung adenocarcinoma cells (A549 cells) were cultured on the scaffolds and then used for the cytotoxicity evaluation of total particulate matter (TPM) in mainstream smoke from two different cigarettes. The evaluation results were compared with those of two-dimensional cell culture systems. The results showed that:1) A549 cells could proliferate and form a cell layer on three-dimensional cell scaffolds. The cells proliferated faster on the scaffolds modified by acrylic acid (Aa). 2) After culturing for 21 days, the optical density (OD) value of A549 cells cultured on the modified three-dimensional cell scaffolds was 3.601, which was higher than that (3.055) of the cells cultured on a two-dimensional plate for 7 days. 3) The TPM in smoke of 3R4F and CM8 cigarettes had a good dose-effect relationship with the viability of A549 cells cultured on two-dimensional plate or three-dimensional cell scaffolds. At high TPM concentrations, the cell viability was higher on the three-dimensional cell scaffolds than on the two-dimensional plate. This study provides a new three-dimensional cell culture method for in vitro toxicology evaluation of cigarette smoke.