This study evaluated the efficacy of the proposed matrix-assisted room-temperature (MART) drying as an alternative to freeze-drying for the thermostabilization of functional proteins in a solid state. To achieve this, protective agents were formulated with functional proteins, and the mixture was dried on a biocompatible cellulose fiber matrix. Drying was carried out at room temperature or elevated temperatures (∼30 °C), either through dry air circulation (MART-DA drying) or under a vacuum (MART-V drying). The entire drying process did not involve any refrigeration or freezing steps. The results demonstrated the successful thermostabilization of lactate dehydrogenase (LDH), fibroblast growth factor-2 (FGF-2), and functional enzymes in reverse transcription loop-mediated isothermal amplification (RT-LAMP) reagents through the use of MART drying. The functional proteins were immobilized and effectively encapsulated in sugar glass films, preserving the proteins’ native structure and functions. The sugar glass films were supported by a low-cost three-dimensional (3D) cellulose fiber matrix. Overall, MART drying offers a simple, fast, low energy-consumption, and cost-effective strategy for drying functional proteins for long-term room-temperature storage.