In this study, we evaluated biohydrogen production of Clostridium butyricum and Rhodopseudomonas palustris by immobilized co-culture. Effects of the free cells and immobilized cells, immobilized biomass ratio, sucrose concentration and initial pH on hydrogen production were investigated. The immobilized co-culture can achieve high cumulative hydrogen (604ml) as compare to free co-culture cumulative hydrogen while the initial sucrose concentration was 17.8g/l. The optimum C. butyricum/R. palustris ratio was 1/10 yielding the highest cumulative hydrogen (728ml). High sucrose concentration (above 35.6g/l) would inhibit hydrogen production. The optimal initial pH for hydrogen production of immobilized co-culture was 7.0 (cumulative hydrogen 830ml).