► CALB has been reversibly immobilized on octyl Sepharose at different rates. ► Different percentages of the amino groups in the enzyme have been modified with glutaraldehyde. ► Rapidly immobilized enzyme presented mainly aggregates after modification. ► Slowly immobilized CALB presented mainly monomers after modification. ► Enzyme activity and stability were increased by the modification. Lipase B from Candida antarctica (CALB) has been immobilized on octyl-agarose in two ways: rapidly, in 5mM sodium phosphate (85% immobilization yield after 30min), or slowly, in the presence of 30% (v/v) ethanol (40% immobilization yield after 30min). Both biocatalysts were treated with glutaraldehyde in order to obtain different modification degrees on their amino groups (25, 50 and 100% modification). SDS-PAGE and detergent desorption experiments showed that, when the immobilization was performed in absence of ethanol, very large aggregates were formed by intermolecular crosslinking, while when 30% ethanol was added during immobilization, almost 90% of the enzyme remained as a monomer. The stability of both derivatives improved upon modification, both in thermal inactivation experiments (at pHs 5, 7 and 9) or in the presence of 50% (v/v) dimethylsulfoxide, achieving stabilization values ranging between 5 and 20 depending on the inactivation conditions. The stability increased proportionally with the modification degree, and was also higher when intermolecular bonds were performed (by a 2–4 factor). Moreover, the activity/pH profile was completely altered after enzyme modification, and, under certain conditions, the activity of the modified biocatalysts doubled that of the non-modified immobilized CALB. Results show that the addition of ethanol permits to have a distance between enzyme molecules that did not allow intermolecular crosslinking, and this has permitted to distinguish between the effects of intramolecular glutaraldehyde modifications and intermolecular glutaraldehyde crosslinking. The simple and controlled treatment of CALB-octyl with glutaraldehyde has proved to be an effective way to obtain a biocatalyst with improved activity and stability under different conditions.