Amino acid arrays comprising bioluminescent amino acid auxotrophic Escherichia coli are effective systems to quantitatively determine multiple amino acids. However, there is a need to develop a method for convenient long-term preservation of the array to enable its practical applications. Here, we reported a potential strategy to efficiently maintain cell viability within the portable array. The method involves immobilization of cells within agarose gel supplemented with an appropriate cryoprotectant in individual wells of a 96-well plate, followed by storage under freezing conditions. Six cryoprotectants, namely dimethyl sulfoxide, glycerol, ethylene glycol, polyethylene glycol, sucrose, and trehalose, were tested in the methionine (Met) auxotroph-based array. Carbohydrate-type cryoprotectants (glycerol, sucrose, and trehalose) efficiently preserved the linearity of determination of Met concentration. In particular, the array with 5% trehalose exhibited the best performance. The Met array with 5% trehalose could determine Met concentration with high linearity (R² value = approximately 0.99) even after storage at −20 °C for up to 3 months. The clinical utilities of the Met and Leu array, preserved at −20 °C for 3 months, were also verified by successfully quantifying Met and Leu in spiked blood serum samples for the diagnosis of the corresponding metabolic diseases. This long-term preservation protocol enables the development of a ready-to-use bioluminescent E. coli-based amino acid array to quantify multiple amino acids and can replace the currently used laborious analytical methods.