The use of immobilized enzymes is mandatory for the easy separation of the enzyme, the unreacted substrates, and the obtained products to allow repeated enzymatic assays without cumbersome purification steps. The immobilization procedure is however critical to obtain a high fraction of active enzyme. In this article, we present an enzyme immobilization strategy based on a catechol functionalized alginate. We demonstrate that alkaline phosphatase (ALP) remains active in multilayered films made with alginate modified with catechol moieties (AlgCat) for long duration, that is, up to 7 weeks, provided the multilayered architecture is crosslinked with sodium periodate. This cross-linking reaction allows to create covalent bonds between the amino groups of ALP and the quinone group carried by the modified alginate. In the absence of cross-linking, the enzymatic activity is rapidly lost and this reduction is mainly due to enzyme desorption. We also show that NaIO4 cross-linked (AlgCat-Alp)(n) films can be freeze-dried and reused at least 3 weeks later without lost in enzymatic activity.