Serratia sp. W4-01 was immobilized in chitosan-activated carbon beads and used for diesel oil removal. The type and concentration of chitosan, activated carbon content, and bead diameter were investigated as factors affecting diesel oil removal. The results showed that 2% (w/v) squid pen chitosan beads modified with 1% activated carbon (w/v) and with a 3-mm diameter had a good spherical shape and strength as well as diesel oil removal capability. The immobilized W4-01 cells removed more than 40% of diesel oil after 7 days when the initial diesel oil concentration was 100 to 400 mg L, whereas 29-36% of diesel oil was removed after 14 days when the initial concentration was 800 to 1000 mg L. Additionally, the immobilized cells maintained the ability to remove diesel oil over a pH range of 5-11. The addition of a biosurfactant increased the diesel oil removal from 62 to 75%. The reusability tests revealed that the ability of immobilized cells to remove diesel oil was enhanced after reuse, and 50-90% of diesel oil was removed during 2 to 12 reuse cycles. The stability and survival of W4-01 cells was confirmed by scanning electron microscopy and confocal laser scanning microscopy. The results of this study showed the potential use of W4-01 cells immobilized in chitosan-activated carbon beads for future applications in remediating diesel contamination.