Phenylketonuria (PKU) is an autosomal recessive disorder caused by a defective phenylalanine hydroxylase (PAH), which catalyzes the hydroxylation of l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr) in presence of the cofactor tetrahydrobiopterin (BH4). Defective PAH causes accumulation of phenylalanine, which has neurotoxic effects and leads to dermatological, behavioral, and neurocognitive problems. Treatments for this disease consist in life-long diets that are hard for patients to keep, or supplementation with BH4. In this study, we propose a system where a probiotic lactic acid bacteria (LAB) can be used as vehicle to express in situ an engineered human PAH. Engineered PAHs contain a secretion peptide, a gastrointestinal signal (GI), the human PAH, and a flexible glycine linker followed by the fluorescence protein mEGFP. Engineered constructs were successfully transformed, expressed, and secreted in Lactobacillus plantarum CM_PUJ411. PAH construct containing either the signal peptide GI1 or GI2 were transported through a Caco-2 cell monolayer. Nevertheless, the one containing GI1 allowed the highest transport through the cell monolayer. Co-culture of L. plantarum and Caco-2 cells showed that engineered PAH is produced in-situ and transported through the cell monolayer. Finally, the activity test showed that the engineered PAH secreted by L. plantarum CM_PUJ411 is active, leading to a reduction in l-Phe and an increase in l-Tyr levels, respectively. These results show the potential of this system as a new therapeutic alternative for the treatment of PKU patients.