Salvianic acid A (SAA) is a catechol compound known for its diverse physiochemical functions and has significant applications in the food and pharmaceutical industries. 4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H) is a critical enzyme for SAA biosynthesis, and improving its activity towards p-hydroxyphenyllactate acid (4HPLA) is essential for highly efficient SAA production in stable biosynthetic pathways. To address this, the distal site and loops of the substrate pocket were modified to improve 4HPA3H catalytic activity towards 4HPLA using computer-aided molecular modification methods. As a result, we identified and mutated two critical sites in EcHpaB, a 4HPA3H monooxygenase from Escherichia coli: T398, a substrate loop site, and M205, a distal site. The mutants M205F, T398S, and M205F/T398S enabled 2.51-, 2.07-, and 2.20-fold increases in catalytic efficiency (k