Pollen in the atmosphere is one of the common bioaerosol materials that can be harmful to human health. To collect and predict the pollen in the atmosphere, a sampling module of a cyclone wet-wall bioaerosol sampler was designed using the computational fluid dynamics (CFD) method and response surface method (RSM). The parabolic shape was chosen for the sampling module based on the axial and tangential velocities of flow in the module. Then, the main diameter of the sampling module was determined. The significance analysis of the main parameters of the sampling module on the capture rate was conducted using the Plackett-Burman (PB) design method. In descending order, the items are inlet width > outlet diameter > inlet height > base diameter > cone height > cylinder height > outlet pipe insertion depth. The test center point of Box-Behnken design (BBD) for the four significant parameters, namely inlet width, outlet diameter, inlet height, and cone height, were determined using the steepest climb test. Then, the optimal parameters of the sampling module were obtained using RSM and the optimal parameters were an inlet height of 15.4 mm, an inlet width of 5.4 mm, an outlet diameter of 14.7 mm, and a base diameter of 13.3 mm. Finally, the simulated capture rate of the sampling module was validated by the experimental results. The capture rate of the optimal sampling module for 2.5 μm PSL particles was 82.93 ± 4.25%. The results of this study can provide a practical method for designing the sampling module using the CFD model and RSM. Additionally, it can provide a high collection rate sampling module for pollen and bioaerosol in the environment. Display omitted •The main parameters of the sampling module on the capture rate were determined.•The optimal parameters of the sampling module were obtained using response surface methodology.•The designed sampling module was verified by the experiments.•The capture rate of the sampling module with optimal parameters was 82.93 ± 4.25%.