The porous structure of scaffold is important parameter for cell and drug delivery. In this study, The chitosan bead scaffolds having open macropores interconnected with micropores were prepared from phase separation process and the effect of quenching rate on pore size of the beads was investigated. The morphology of the beads was analyzed by scanning electron microscopy(SEM). The addition of non-solvent in chitosan solution was to make the homogeneous micropores and improved interconnectivity between macropores without any surface skin layer. The macropore size of the beads was controlled to change of temperature of cooling medium. The macropore size of the new prepared chitosan bead scaffolds(1CS-RT70 and 10B-RT70) was the largest of the chitosan bead scaffolds fabricated from conventional method. Chondrocytes isolated from rabbit costal cartilage were cultured on the beads. Cellular viability and proliferation were assayed by cell counting kit- 8. Initial cell proliferation in the beads with micropores was higher than that in the beads without micropores. Cell attachment and distribution in the beads were also analyzed by SEM and Safrain-O staining after 14 days culture. The proliferation of chondrocytes were exhibited in only surface of the 1CS-LN2 and 10B-LN2 bead scaffolds but exhibited in both surface and center of the 1CS-RT70 and 10B-RT70 bead scaffolds. Therefore the new prepared chitosan bead scaffolds are more suitable for tissue engineering application.