Chitosan was derivatized by two methodologies for analyzing their effect on chitosan physicochemical characteristics and its applicability as carrier for Bacillus circulans β-galactosidase immobilization. Glutaraldehyde (GA) and epichlorohydrin (EPI) were used for crosslinking and activation of chitosan, producing the corresponding supports (C-GA and C-EPI-EPI) after a one-step and a two-step process, respectively. The spherical shape and mean diameter of chitosan particles was not significantly affected by polymer derivatization, while Fourier transform infrared analysis showed that in both cases, chitosan polymer was chemically modified. TGA analysis indicated that C-EPI-EPI was the most thermally stable. The high degree of activation of C-EPI-EPI (586 μmol of aldehydes/g) resulted in the highest loss of activity during immobilization; hence a support with 100 μmol of aldehydes/g was produced (C-EPI-EPI100). The highest expressed activity (89.3 IU/g) was obtained with the enzyme immobilized in C-GA, while the biocatalyst with highest thermal stability at 60°C was obtained with C-EPI-EPI100 (half-life was 84-fold higher than the one of the soluble enzyme). The best compromise between biocatalyst expressed activity and thermal stability corresponded to β-galactosidase immobilized in C-EPI-EPI100. According to this study, chitosan derivatized with EPI is a thermally stable carrier appropriate for producing highly stable immobilized B. circulans β-galactosidase. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40171.