Lactococcus lactis-expressing phenylalanine ammonia-lyase (LLEP) has been used to treat one of the classic genetic diseases, phenylketonuria (PKU). However, the action of stomach fluid and short residence time of LLEP at the site of absorption become the "neck" for the oral administration of LLEP. To solve these problems, pH-sensitive Ca-alginate microparticles designed as an oral administration carrier were prepared by a spray-solidification method in this study. The spray conditions influenced the size of the Ca-alginate micropartides; thus, conditions were optimized to obtain microparticles with smaller particle size for oral administration to mice. Subsequently, LLEP was encapsulated into Ca-alginate microparticles and the activity retention of LLEP released from the micropartides was examined after the microparticles passed through simulated gastric fluid. The results showed that LLEP could be well protected against simulated gastric fluid and that the final activity retention was up to 92.9%. The effects of alginate concentration on the release profile in vitro and the encapsulation efficiency (EE) were studied, and the results revealed that the microparticles possessed the highest EE and a reasonable release rate when the alginate concentration was 1.0 wt %. This alginate concentration, combined with optimized spray conditions, was used to prepare LLEP-encapsulated microparticles, and they were administered orally to mice with phenylketonuria (PKU). Compared with the groups given blank microparticles and non-encapsulated LLEP, the increase of the blood phenylalanine (Phe) level was significantly slowed after a 7 day treatment with LLEP-encapsulated microparticles. Consequently, the Ca-alginate microparticle developed by the spray-solidification method is a promising carrier of LLEP for oral administration.