Single-walled carbon nanotubes (SWCNTs) synthesized by the improved arc discharge method were modified with a fluorocarbon chain, and the maintenance of trypsin activity upon adsorption from the subphase to the interfacial monolayer of SWCNTs was evaluated. The adsorption of trypsin on the fluorocarbon-modified SWCNT monolayer was confirmed by morphological and spectroscopic evaluations. Fiber morphology studies revealed that the fluorocarbon-modified SWCNT monolayer was covered by trypsin, and a trypsin-derived amide band was detected in the infrared spectra of the multilayers. After adsorption onto the fluorocarbon-modified SWCNT film, the ability of trypsin to cleave the fluorescent casein chain was maintained even at 160 °C. Furthermore, circular dichroism (CD) spectra showed that the second-order structure of the activity of trypsin adsorbed on the fluorocarbon-modified SWCNT was maintained up to nearly 200 °C. At 200 °C, the enhancement of emission intensity by casein chain cleavage was negligible, and the CD signal resulting from the negative Cotton effect was completely altered at 250 °C.