The effect of carbonic anhydrase (CA) on the separation performance of thin poly(amidoamine) (PAMAM) dendrimer/poly(ethylene glycol) (PEG) hybrid membranes was investigated. CA, a type of enzyme, was used to promote CO2 hydration and dehydration reactions and to assess whether these reactions were the rate-limiting step in CO2 permeation through the membrane. The relationship between the membrane thickness and the CO2 permeance was evaluated in CO2/H2 or CO2/He separation using PAMAM/PEG hybrid membranes (thickness: 10–100 μm) with and without CA. Without CA, the CO2 permeance of PAMAM/PEG hybrid membranes was not inversely proportional to the membrane thickness. On the other hand, with CA, the CO2 permeance was inversely proportional to the membrane thickness. It was implied that, without CA, the rate-limiting step of CO2 transport was either the CO2 hydration reaction at the feed side or the CO2 dehydration reaction at the permeate side. On the other hand, with CA addition, the rate-limiting step of CO2 transport was diffusion, and CO2 permeance could be increased without sacrificing the selectivity by reducing membrane thickness. The effect of the position of CA (i.e., on the surface and/or reverse surface) on CO2 separation performance was investigated to evaluate which reaction was the rate-limiting step of CO2 permeation through the membrane. It was suggested that the rate-limiting step of CO2 permeation was CO2 dehydration reaction at the permeate side.