Burdock fructooligosaccharide (BFO) was carboxymethylated by aqueous method to prepare modified products (CM-BFO) with different degrees of carboxymethyl substitution (DS). The relationship between reaction conditions and DS and the interaction of various factors were investigated by single factor and response tests. The structures of BFO and CM-BFO were characterized by GC,FTIR, 1HNMR, and 13CNMR. RAW264.7 cells were used as a model to test the immune activity of CM-BFO with different DS in vitro. The results showed that 0.5 g of BFO was alkalized with 30 mL of 20% NaOH aqueous solution for 1 h, 30 mL of 2.5% chloroacetic acid aqueous solution was used as carboxymethylation reagent, the reaction temperature was 65 ℃, the reaction time was 3.3 h, and the highest ds of BFO carboxymethylation was 1.06 under the optimal conditions; Both BFO and CM-BFO could enhance the immune activity of RAW264.7 cells. The immune activity of three kinds of CM-BFO with DS of 0.82, 0.93 and 0.96 was significantly increased compared with BFO, while the CM-BFO with the lowest (0.73) and highest DS (1.06) was slightly increased. The immune activity of CM-BFO increased first and then decreased with the increase of DS in a certain range.