This paper studies a kind of hollow nanospheres prepared by self‐assembly β‐cyclodextrins (β‐CDs) and poly(ethylene oxide)‐poly(propylene oxide)‐poly(ethylene oxide) (pluronic F127) for gene delivery. It was found that this kind of hollow nanospheres enable load PEI10K/DNA and the resulting F127? NH2? βCD/(PEI10K/DNA) with 0.08 µg/well DNA display equal or higher gene delivery capability compared to PEI10K/DNA with 1 µg/well DNA in the absence or presence of serum. The cytotoxicity of the nanospheres was over 100 times lower than that of PEI10K.