Controlled β-cyclodextrin (β-CD) core-based cationic star polymers have attracted considerable attention as non-viral gene carriers. Atom transfer radical polymerization (ATRP) could be readily used to produce the star-shaped polymers. The precise control of the number of initiation sites on the multifunctional core was of crucial importance to the investigation of the structure–property relationship of the functional star gene carriers. Herein, the controlled multiarm star polymers consisting of a β-CD core and various arm lengths of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) were prepared via ATRP from the chloroacetylated β-CD with well-designed initiation sites. Generally, these star polycations can condense plasmid DNA into 100–150nm nanoparticles with positive zeta potentials of 30–40mV at N/P ratios (star polymer to DNA ratios) of 17 or higher. The effects of arm numbers and lengths on gene delivery were investigated in detail. With a fixed length of the PDMAEMA arm, the fewer the number of arms, the lower the toxicity. The star polycations with suitable arm numbers possess the best transfection ability. On the other hand, with the fixed molecular weights, the shorter the arms, the lower the toxicity. The polymers with 21 arms possess the lowest transfection efficiency.