Strategies that speed up the on-command release of proteins (e.g., enzymes) from stimuli-responsive materials are intrinsically necessary for biosensing applications, such as point-of-care testing, as they will achieve fast readouts with catalytic signal-amplification. However, current systems are challenging to work with because they usually exhibit response times on the order of hours up to days. Herein, we report on the first effort to construct a fast-responding gating system using protein-encapsulating functional DNA superstructures (denoted as protein@3D DNA). Proteins were directly embedded into 3D DNA during the one-pot rolling circle amplification process. We found that the specific DNA-DNA interaction and aptamer-ligand interaction could act as general protocols to release the loaded proteins from 3D DNA. The resulting gating system exhibits fast release kinetics on the order of minutes. Taking advantage of this finding, we designed a simple paper device by employing protein@3D DNA for colorimetric detection of toxin B (Clostridium difficile marker). This device is capable of detecting 0.1 nM toxin B within 16 minutes.