Free-standing silk films are useful materials to manufacture nanopatterned optical elements and to immobilize bio-dopants such as enzymes while maintaining their biological activity. These traits were combined by incorporating hemoglobin into free-standing silk diffraction gratings to fabricate chemically responsive optofluidic devices responsive to ambient gas conditions, constituting a simple oxygen sensor. This type of self-analyzing optical system is enabled by the unique ability to reproduce high-fidelity optical structures in silk while maintaining the activity of entrapped proteins such as hemoglobin. These bioactive optical devices offer a direct readout capability, adding utility into the bioresponsive material arena. © 2009 American Institute of Physics.