This study aimed to improve the production of β-cyclodextrin (β-CD) by microbial cells immobilized on synthetic or loofa sponges both with and without the use of alginate or chitosan. The most suitable matrix for the immobilization of Bacillus firmus strain 7B was synthetic sponge and for Bacillus sphaericus strain 41 was loofa sponge. After 330 days of storage, the β-CD production by Bacillus firmus and Bacillus sphaericus remained at around 41% and 49%, respectively, of initial levels. After 24 days of immobilization on loofa sponge, Bacillus sphaericus strain 41 achieved an improved operational stability, reaching 86.6 mM β-CD after 20 days of production, compared to only 32.8 mM of β-CD produced by free Bacillus sphaericus strain 41 cells. The expected increase in β-CD production by immobilized cells of Bacillus firmus strain 7B on synthetic sponge for 4 days was not statistically different to that for cells immobilized for 24 days. The application of this process on an industrial scale using loofa sponge, an inexpensive and renewable matrix, will allow the stable production of β-CD.