Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease caused by deficiency of α-l-iduronidase (IDUA), which results in the lysosomal accumulation of glycosaminoglycans (GAG) leading to widespread clinical manifestations. The microencapsulation of IDUA overexpressing recombinant cells has been considered as a promising strategy for the treatment of MPS I. This study aimed at the optimization of alginate microcapsules containing recombinant BHK (Baby Hamster Kidney) cells (rBHK) overexpressing IDUA produced by electrostatic extrusion technique. The alginate microcapsule (MC-A) optimization study was carried out by means of an experimental Box-Behnken Design that allowed the simultaneous evaluation of the influence of voltage (kV), alginate/cell suspension flow (mL/h), and alginate concentration (%) on size and IDUA activity. The optimal conditions of voltage (10 kV), flow (25 mL/h), and alginate concentration (1.3%) made possible to obtain the smallest microcapsules showing the highest IDUA activity. After optimization, the microcapsules were sequentially coated with PLL and alginate (MC-APA) to increase their stability. MC-A and MC-APA presented monodisperse populations (span < 1.22) with an average diameter of less than 350 μm. The coating increased the mechanical stability of MC-APA by about 6-fold and modulated the permeability to the enzyme. Surface analyzes of MC-APA showed the presence of PLL bands, suggesting that the last alginate layer appears to have only partially coated the PLL. After 30 days of subcutaneous implantation of the MC-APA microcapsules containing rBHK cells in a MPS I murine model, a significant increase in IDUA activity was observed in the skin near the implant. Histological analysis revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the conditions evaluated. Taken together, the overall results demonstrate the feasibility of MC-APA as a potential alternative for local treatment of MPS I.