Papain, a member of the cysteine protease family, is an endopeptidase extracted from papaya latex (Carica papaya), which finds wide application in food technology. Many strategies have been researched for greater efficiency and yield of the enzymatic hydrolysis processes, such as the inclusion of an organic solvent in the reaction medium (acetonitrile, among others) or the use of this enzyme immobilized. The immobilization of enzymes in solid supports has been finding more applications in the different processes, bringing several advantages to the enzyme that will be immobilized, such as greater stability, possibility of reuse, ease of removal of the reaction medium and even the protection of its protein chain. In this work the use of organic solvent in the papain activity, free and immobilized in support of glyoxyl agarose 4BCL and 10BCL, was tested. The enzyme activity was determined with the use of the benzoyl-DL-arginine-p-nitroanilide substrate (BAPNA) in pH 7.0 buffer. The enzyme was immobilized at different times to check its stability to acetonitrile. Although we obtained a total immobilization of 100% of the available load (8 mg papain/g support), the recovered activity was 48.7%. For 10BCL support the immobilization was of 86% with recovery activity of 13.9%. The 4BCL support was chosen for the other assays. The enzymes immobilized for 24 hours were more stable, maintaining 52% of the activity in 30% of acetonitrile, and increasing activity (127% in comparison to free papain) when the acetonitrile concentration was 20%. As expected, it had a lower stability, maintaining only about 45% in 20% of acetonitrile and 26% when the acetonitrile concentration was 30% (when free papain maintain about 15%). The results are promising and indicate that immobilization may increase the stability of papain in medium containing acetonitrile which at low concentrations may even provide an increase in its activity.